.■■ltfii:ii[Liiitut:::.i!lJl 


•f 


Demonstrating  phagocytosis  of  various  bacteria  as  observed  in  the  determina- 
tion of  the  opsonic  index.  1,  Staphylococcus;  2,  Micrococcus  candicans;  3,  Strepto- 
coccus pyogenes;  4,  Colon  bacillus;  5,  Gonococcus;  6,  Tubercle  bacillus.  {See  p.  234.) 


APPLIED 

IMMUNOLOGY 

THE  PRACTICAL  APPLICATION  OF  SERA 
AND  BACTERINS  PROPHYLACTICALLY, 
DLVGNOSTICALLY  AND  THERAPEUTICALLY 


WITH  AN  APPENDIX  ON 

SERUM  TREATMENT  OF  HEMORRHAGE,  ORGANOTHERAPY 
AND  CHEMOTHERAPY 


BY 

B.  A.  THOMAS,  A.M.,  M.D. 

PROFESSOR  OF  GENITO-URINART  SURGERY  IN  THE  POLYCLINIC  HOSPITAL  AND  COLLEGE 

FOR  GRADUATES  IN  MEDICINE  ;   INSTRUCTOR  IN  SURGERY  IN  THE  UNIVEK81TT 

OF   PENNSYLVANIA  ;   ASSOCIATE  IN  THE  WILLIAM   PEPPER 

LABORATORY  OF  CLINICAL  MEDICINE 

AND 

R.  H.  IVY,  M.D.,  D.D.S. 

ASSISTANT  INSTRUCTOR  IN  SURGERY  IN  THE  UNIVERSITY  OP  PENNSYLVANIA  ;  INSTRUC- 
TOR IN  GENITO-URINARY  SURGERY  IN  THE  POLYCLINIC  HOSPITAL  AND 
COLLEGE  FOR   GRADUATES   IN   MEDICINE 
PHILADELPHIA 


5  COLORED  INSERTS  AND  68  ILLUSTRATIONS  IN  TEXT 


PHILADELPHIA  AND  LONDON 

B.  LIPPINCOTT  COMPANY 


T5 


BiOtOGX 


Copyright,  1915 
By  J.  B.  LiPPiNcoTT  Company 


Printed  by  J.  B.  Lippincott  Company 
The  Washington  Square  Press,  Philadelphia,  U,  S.  A. 


To 

ALFRED  STENGEL.  M.D. 

PROFESSOR  OF  MEDICINE  IN  THE  UNIVERSITY  OF  PENNSTLVANIA 

IN  GRATEFUL  RECOGNITION  OF  HIS  WISE  COUN- 
SEL AND  KINDLY  ADVICE  THIS  VOLUME  IS 
BESPECTFULLY    DEDICATED    BY    THE    AUTHOBS 


Sf)Kf^.^i 


PREFACE 

Despite  the  enormous  strides,  experimentally  and 
clinically,  that  have  marked  the  progress  in  serological 
and  bacteriological  research  in  medicine  for  the  past 
quarter  of  a  century,  leading  to  results  of  the  highest 
clinical  value  diagnostically  and  therapeutically,  three 
facts  stand  out  in  bold  relief:  First,  that  the  average 
practitioner's  knowledge  of  biological  therapeusis  is  a 
"  dangerous  thing  "  because  he  does  not  "  drink  deep 
of  the  Pierian  spring  " ;  second,  insufficient  practical 
instruction  is  given  to  students  in  our  medical  schools 
in  view  of  the  prevalent  practice  of  this  mode  of  diag- 
nosis and  treatment;  third,  authors  have  sadly  neg- 
lected to  give  a  concise  presentation  of  immunology  in 
its  entirety  for  the  practical  grasp  and  comprehension 
of  students  and  practitioners.  The  vast  majority  of 
standard  works  on  the  subject  of  "  immunity  "  are 
exhaustive  treatises  on  the  experimental  and  theoreti- 
cal phases  of  the  subject  and  are  often  unintelligible 
and  of  little  value  to  the  average  practicing  physician. 

The  result  of  the  above  has  been  that  pharmaceuti- 
cal firms  have  assumed  the  function  of  the  Department 
of  Therapeutics  in  our  medical  colleges,  not  always 
impartially  and  to  the  best  interests  of  medical  science. 


vi  PREFACE 

Obviously,  licentiates  to  practice  medicine  should  re- 
ceive instruction  of  a  definite  and  practical  nature  in 
this  important  branch  of  modern  medicine,  or  immu- 
nology should  receive  a  place  in  the  classification  of 
specialties,  a  fact  which  the  importance  of  the  subject 
may  command. 

In  this  book  it  has  been  the  aim  of  the  authors 
purposely  to  omit  most  of  the  experimental  research 
and  to  present  theories  only  in  so  far  as  they  may 
assist  in  a  more  thorough  comprehension  of  biological 
prophylaxis,  diagnosis  and  therapeusis. 

The  primary  object  has  been  to  crystallize  and  de- 
tail the  practical  phases  of  serum  and  bacterin  applica- 
tions in  medicine,  thereby  enabling  the  student  and 
general  practitioner,  with  even  a  slight  laboratory  ex- 
perience, to  appreciate  the  significance  of,  and  more 
competently  apply  the  principles  underlying,  immu- 
nology. In  order  to  render  the  treatise  more  complete 
allusion  has  been  made  in  places  to  certain  allied  sub- 
stances that  have  been  utilized  from  time  to  time  in 
attempts  at  immunization,  with  a  consideration  of 
their  merits  and  demerits. 

The  interest  and  close  association  of  blood  trans- 
fusion, organotherapy  and  administration  of  salvarsan 
and  neosalvarsan  to  the  main  subject  have  prompted 
the  authors  to  devote  an  appendix  to  their  discussion. 

The  reader  who  may  have  his  enthusiasm  aroused 


PREFACE  vii 

and  is  ambitious  to  enter  more  deeply  into  the  realms 
of  immunity  is  referred  to  the  systems  of  Kolle  and 
Wassermann,  and  Kraus  and  Levaditi,  or  the  works 
of  Ehrlich  and  Bolduan,  and  Bordet  and  Gay  on 
"  Studies  in  Immunity  ";  Emery  on  "  Immunity  and 
Specific  Therapy  ";  Wright  on  "  Studies  on  Immuni- 
zation ";  Simon  on  "  Infection  and  Immunity  ";  Cit- 
ron on  "  Immunity,"  and  Vaughan  on  "  The  Relation 
of  Anaphylaxis  to  Immunity  and  Disease." 

B.  A.  Thomas 
R.  H.  Ivy 

William  I*epper  Laboratory  of 
Clinical,  Medicine, 
Philadelphia,  1915 


CONTENTS 


CHAPTER  I  PAOB 

Introduction 1 

Immunity  and  Immunization — Natural  and  Acquired  Immunity — 
Active  and  Passive  Immunization — Mechanism  of  the  Production  of 
Immunity — History  and  Development  of  Immunology. 

CHAPTER  II 

Antigens  and  Antibodies 19 

Toxins  and  Antitoxins — Agglutinins — Precipitins — Lysins — Isocytoly- 
sins — Opsonins — Antiferments — Auto-antibodies — Allergy  and  Ana- 
phylaxis. 

CHAPTER  III 
Ehrlich's  Side-chain  Theory 24 

CHAPTER  IV 
Anaphylaxis  or  Hypersusceptibility 27 

CHAPTER  V 

Antisera 34 

Preparation  of  Antisera — Indications  for  Therapeutic  Use  of  Antisera. 

CHAPTER  VI 

Antitoxic  Sera 42 

Antidiphtheritic  Serum — Antitetanic  Serum — Antigonococcic  Serum — 
Antituberculosis  Serum — Antidysenteric  Serum — Antibotulism  Serum 
— Antiphytotoxic  Serum — Antivenin. 

CHAPTER  VII 

Antibacterial  Sera 55 

Antistaphylococcic  Serum — Antistreptococcic  Serum — Antipneumo- 
coccic  Serum — Antigonococcic  Serum — Antimeningococcic  Serum — 
Antityphoid  Serum — Anticolonic  '  Serum — Antidysenteric  Serum — 
Anticholera  Serum — Antiplague  Serum — Anti-anthrax  Serum — Anti- 
melitensic  Serum. 

CHAPTER  VIII 

Miscellaneous  Sera  and  Extracts 64 

Antirabic  Serum — Antileprosy  Serum — Antityphoid  Extract  of  Jez — 
Leucocytic  Extract — Antiferment — Anticarcinomatous  Extracts — 
Pyocyanase — Antithyroid  Serum  and  Extracts — Spangler's  Crotalin 
— Phylacogens. 

CHAPTER  IX 

Agglutinins 72 

The  Widal  Phenomenon  and  Other  Agglutination  Reactions. 

ix 


X  CONTENTS 

CHAPTER  X 

Precipitins 82 

Significance  and  Application  of  Precipitins — Technic  of  Reaction — 
Specific  Identification  of  Blood  and  Other  Proteins. 

CHAPTER  XI 

Ltsins 86 

Bacteriolysins  and  Cytolysins  (Hsemolysins). 

CHAPTER  XII 

Fixation  of  Complement 90 

Principles  of  the  Reaction — Bordet-Gengou  Phenomenon — Wasser- 
mann-Neisser-Bruck  Modification — Technic  of  the  Wassermann  Re- 
action in  the  Diagnosis  of  Syphilis — Modifications  of  the  Wassermann 
Reaction — Hecht-Weinberg  Modification — Clinical  Application  of  the 
Wassermann  Reaction — Effects  of  Treatment  on  the  Wassermann 
Reaction. 

CHAPTER  XIII 

Fixation  of  Complement  (Continued) 141 

Gonococcus  Complement-fixation  Test — Serum  Diagnosis  of  Echino- 
coccus  Disease — Complement-fixation  Reaction  in  Typhoid  Fever — 
Complement-fixation  Reaction  in  Tuberculosis — The  Complement- 
fixation  Reaction  as  Applied  in  Protein  Differentiation  (Neisser-Sachs 
Reaction). 

CHAPTER  XIV 

Miscellaneous  Biochemical  Reactions 157 

Abderhalden's  Biological  Test  for  Pregnancy — Sero-enzyme  Test  for 
Syphilis — Abderhalden-Fauser  Reaction  in  Mental  Diseases — Meio- 
stagmin  Reaction — Epiphanin  Reaction. 

CHAPTER  XV 

SPEaFic  Bacterial  Cutaneous  Reactions 167 

Allergic  Phenomena — Tuberculin  Tests — Luetin,  Gonorrhoea!  and 
Typhoid  Tests — Schick's  Diphtheria  Toxin  Skin  Reaction. 

CHAPTER  XVI 

Tuberculin  Therapy 193 

Prophylaxis — Therapeutic  Administration  of  Tuberculin — Available 
Preparations — Modes  of  Administration  and  Dosage — Control  of 
Tuberculin  Treatment — Limitations  and  Contra-indications — Indica- 
tions and  Results. 

CHAPTER  XVII 
Phagocytosis 211 

CHAPTER  XVIII 
Recovery  from  Bacterial  Infections 21S 


CONTENTS  xi 

CHAPTER  XIX 

Bacterial  Inoculation 216 

Principles  Underlying  Inoculation  Therapy — Preparation  of  Bacterins 
— Autogenous  versus  Heterogeneous  Preparations — Clinical  Symptoms 
versus  Opsonic  Index  in  Control  of  Treatment. 

CHAPTER  XX 

The  Opsonic  Index 228 

Definition  of  Opsonins  and  the  Opsonic  Index — Technic  of  Determina- 
tion of  the  Opsonic  Index — Interpretation,  Value  and  Limitations  of 
the  Opsonic  Index. 

CHAPTER  XXI 

Practical   Application   of    Bacterial    Inoculation    in   Medicine, 

Prophylactically  and  Therapeutically 242 

General  Considerations — Induced  Auto-inoculation — Duration  of  Ac- 
tive Immunity — Modes  and  Technic  of  Administration  of  Bacterins — 
Dosage — Contra-indications,  Limitations  and  Causes  of  Failure  in 
Bacterin  Therapy — Application  and  Results  of  Bacterial  Inoculations 
in  Special  Diseases — Diseases  of  the  Skin  and  Soft  Parts — Diseases  of 
the  Genito-urinary  System — Diseases  of  Bones  and  Joints — Diseases 
of  the  Eye,  Ear,  Nose  and  Throat — Diseases  of  the  Lungs — Diseases 
of  the  Alimentary  System — Diseases  of  the  Cardiovascular,  Lymphatic 
and  Nervous  Systems,  also  Other  Acute  Specific  Fevers — Malignant 
Neoplasmata — Yeast  and  Sour  Milk. 

APPENDIX 

Part  A.     Serum  Treatment  of  Hemorrhage 306 

Normal  Fresh  Serum — Precipitated  Horse  Serum — Transfusion  of 
Blood. 

Part  B.     Organotherapy 312 

Thyroid  Gland — Adrenal  Gland — Pituitary  Body — Ovary — Corpus 
Luteum — Thymus  Gland. 

Part  C.     Chemotherapy 318 

Administration  of  Salvarsan  and  Neosalvarsan,  Intravenously,  Intra- 
muscularly and  Intraspinally — Autosalvarsanized  and  Artificially  Sal- 
varsanized  Serum. 


ILLUSTRATIONS 

PLATES 

PAGE 

I.  Demonstrating  Phagocytosis  of  Various  Bacteria  as  Observed  in 

the  Determination  of  the  Opsonic  Index Frontispiece 

II  Graphic  Portrayal  of  the  "Wassermann  Reaction/'  Demonstrating 
the  Results  in  the  Case  to  be  Tested,  the  Positive  Control,  and 
the  Negative  Control 125 

III.  Von  Pirquet's  Cutaneous  Tuberculin  Test  (Positive  Reaction) . .  182 

IV.  Luetin  Cutaneous  Reaction,  Demonstrating  the  Papular  Charac- 

ter of  the  Reaction  on  an  Erythematous,  Indurated  Base 187 

V.  Gonorrhoeal  Allergic  Reaction,  Demonstrating  the  Papulo-erythe- 
matous  Cutaneous  Response  on  the  Third  Day  after  the  Intra- 
dermic  Injection  of  One  Cubic  Millimetre  of  a  Killed  Polyvalent 
Suspension  of  Gonococci 189 

FIGURES 

FIG. 

1.  Diagrammatic  Representation  of  Structure  of  Different  Antibodies    25 

2.  Illustrative  of  Opsonic  Curve,  Showing  Immunity  of  Rabbit 39 

3.  Illustrative  of  Inoculations  and  Curve  of  Opsonic  Indices  Demon- 

strating Immunity  of  Ram 40 

4.  Capillary  Teat  Pipette  for  Removal  of  Senun  from  Clotted  Speci- 

men of  Blood 74 

5.  Widal's  Test,  Positive 78 

6.  Widal's  Test,  Negative 78 

7.  Method  of  Obtaining  Blood  from  Sheep's  Ear 105 

8.  Showing  Method  of  Intravenous  Injection  or  Immunization  of 

Rabbit 107 

9.  Demonstrating  Method  of  Obtaining  Complement  by  Bleeding 

to  Death  an  Anaesthetized  Guinea-pig 109 

10.  Titration  of  Antigen 113 

11.  Showing  Method  of  Collecting  Blood  from  Vein  of  Arm  with 

Keidel's  Vacuum  Ampoule 114 

12.  Showing  Authors'  Method  of  Obtaining  Blood  for  Complement- 

fixation  Reactions 115 

13.  Titration  of  Complement 119 

14.  Showing  Arrangement  of  Tubes  in  Performance  of  Wassermann 

Reaction  on  One  Unknown  Case,  with  Positive  and  Negative 

Controls 126 

ziii 


xiv  ILLUSTRATIONS 

15.  Tuberculous  Mastoiditis 177 

16.  Bilateral  Tuberculous  Epididymitis 178 

17.  All-glass  Tuberculin  Syringe,  Graduated  into  Hundredths  of  a 

Cubic  Centimetre 179 

18.  Chronic  Pulmonary  Tuberculosis 207 

19.  Tuberculous  Coxitis 208 

20.  Electrical  Mechanical  Shaker 219 

21.  Various  Forms  of  Containers  for  Storage  of  Bacterins 223 

22.  Capillary  Glass  Capsules  for  Collection  of  Specimens  of  Blood. .  .  231 

23.  Showing  the  Collection  of  Blood  in  Sodium  Citrate  Saline  Solution  231 

24.  Blood  after  Centrifugation  in  Decalcifying  Medium 231 

25.  Electric  Centrifuge 231 

26.  Various   Ingredients   Necessary   for   the   Determination   of   the 

Opsonic  Index 231 

27.  Washing  the  Culture  of  the  Given  Bacterium  from  the  Culture 

Medium 232 

28.  Opsonizing  Capillary  Pipettes 233 

29.  Opsonizer  or  Thermostat 233 

30.  Illustrating  the  Construction  of  Kuhnhardt's  Spreader 233 

31.  Kuhnhardt's  Spreader  Property  Held  for  Preparation  of  a  Satis- 

factory Smear 234 

32.  Analysis  of  Curve  of  Opsonic  Indices 236 

33.  Case  of  Gonorrhoea!  Arthritis  of  Knee 238 

34.  Pneumonia 239 

35.  Sho^dng  Effect  of  Variable  Concentrations  of  Bacterial  Suspension 

on  Determinations  of  Opsonic  Index 240 

36.  Case  of  Gonococcal  Arthritis 245 

37.  Case  of  Tuberculous  Cervical  Lymphadenitis 246 

38.  All-glass  Hypodermic  Syringe 249 

39.  Case  of  Long-standing  and  Obstinate  Pustular  Acne  Vulgaris 260 

40.  Furunculosis  of  Nostril 261 

41.  Carbuncle  of  Neck 262 

42.  Case  of  Typhoid  Fever 263 

43.  Burns  One-third  to  One-half  Body  Surface;  Multiple  Subcutaneous 

Abscesses 263 

44.  Recurrent  Erysipelas 264 

45.  Tonsiintis,  Peritonsillitis  and  Toxic  Arthritis 265 

46.  Temperature.  Subdiaphragmatic  Abscess  Drained  per  Laparotomy  266 

47.  Pelvic  Abscess  with  Recto-urethro-vaginal  Fistulse 267 

48.  Pyonephrosis 272 

49.  Reno-lumbar  Fistula  Following  Nephrohthotomy  Complicated  by 

Pyonephrosis 273 

50.  Pyelitis  and  Cystitis 274 


ILLUSTRATIONS  xv 

51.  Cystitis  and  Toxic  Neuritis 275 

52.  Chronic  Prostatitis 277 

53.  Typhoid  Fever  Complicated  by  Epididymitis 278 

54.  Acute  Osteomyelitis  of  Tibia  Followed  by  Septicaemia 281 

55.  Corneal  Ulcer  with  Hypopyon 285 

56.  Cultures  both  from  Ethmoidal  Sinuses  and  Bronchial  Expectoration  287 

57.  Illustrating  Typhoid  Fever  Rates  in  United  States  Army 296 

58.  Antityphoid  Inoculation  or  Immunization 297 

59.  Site  for  Deep  Intramuscular  Injection 330 

60.  Position  of  Patient  for  Intravenous  Injection  of  Salvarsan 332 

61.  Apparatus  Ready  for  Preparation  of  Salvarsan  or  Neosalvarsan . .  333 

62.  Thomas'  Salvarsan  and  Neosalvarsan  Outfit 333 

63.  Water  Still  as  Used  in  Authors'  Offices 333 

64.  Illustrating  Method  of  Eliminating  Air  from  Tubing 335 

65.  Thomas'  Salvarsan  and  Neosalvarsan  Burette 335 

66.  Showing  Position  of  Patient  for  Spinal  Puncture 341 

67.  Lumbar  Puncture  with  Strauss  Needle 341 

68.  Intraspinal  Administration  of  Serum,  Using  Syringe 341 


APPLIED  IMMUNOLOGY 


INTRODUCTION 

IMMUNITY  AND  IMMUNIZATION— NATURAL  AND  AC- 
QUIRED IMMUNITY— ACTIVE  AND  PASSIVE  IMMUNIZA- 
TION—MECHANISM OF  THE  PRODUCTION  OF  IMMU- 
NITY—HISTORY AND  DEVELOPMENT  OF  IMMUNOLOGY 

Definition  of  Immunity, — Immunity  is  the  resis- 
tance manifested  by  man  and  various  animal  species  to 
infectious  microorganisms  or  other  foreign  proteins. 
It  is  influenced  by  numerous  factors,  as  changed  en- 
vironment, physical  condition  of  the  animal,  species, 
idiosyncrasies,  virulence  of  the  prevalent  microbe,  etc. 
Conversely,  the  absence  of  this  resistance  implies 
susceptibility.  Occasionally,  hypersusceptibility  to 
certain  proteins  is  observed  and  to  this  state  of 
supersensitiveness  Richet  has  applied  the  term  "  ana- 
phylaxis "  (see  Chapter  IV). 

Two  kinds  of  immunity  are  recognized,  natural 
and  acquired. 

Natural  Immunity. — The  natural  or  spontaneous 
resistance  of  the  animal  organism  to  disease  is  only 

relative,  never  absolute.     The  ability  of  animals  to 

1 


2- •:*•'•'•  '•    APPLIED  IMMUNOLOGY 

ward  off  disease  varies  with  different  species  and 
among  individuals  of  the  same  family.  Under  nor- 
mal conditions,  an  animal  may  be  protected  indefi- 
nitely from  infection.  Nevertheless,  if  his  vital  resis- 
tance be  permitted  to  fall  or  he  be  exposed  to  a  viru- 
lent infection,  his  defences  may  crumble  instantly  and 
disease  be  contracted.  On  the  other  hand,  the  natural 
immunity  of  certain  species  to  infection  is  remarkable ; 
the  negro  to  yellow  fever  and  most  of  the  lower  ani- 
mals to  the  venereal  diseases. 

In  this  connection  allusion  should  be  made  to  local 
immunity.  By  this  we  mean  a  natural  state  of  certain 
organs  or  tissues,  prevalent  from  birth,  due  to  "  in- 
fective tolerance."  For  example,  the  mouth  and  an- 
terior urethra  normally  harbor  many  different  patho- 
genic bacteria  without  ill  effect,  owing  to  life-long 
local  tolerance  with  resultant  immunity.  Introduce 
some  of  these  germs  into  the  synovial  membrane  of  a 
joint  or  into  the  peritoneal  cavity  and  a  virulent  in- 
fection results.  Again,  the  intestinal  tract  tolerates 
colon  bacilli  normally  in  numbers,  which,  if  access  be 
gained  to  the  urinary  tract,  may  precipitate  a  grave 
pathological  process. 

Infections  common  in  warm-blooded  are  rare 
among  cold-blooded  animals  and  vice  versa.  Verte- 
brates and  invertebrates  are  not  subject  to  similar  in- 
fections.    Field   mice   are   susceptible   to   glanders. 


INTRODUCTION  3 

while  house  mice  are  immune.  Tuberculosis  is  more 
prevalent  among  Jersey  than  Holstein  cattle.  Birds 
and  reptiles  are  not  necessarily  subject  to  the  same 
diseases  that  victimize  man.  Thus  the  problem  of 
natural  immunity  presents  many  interesting  phases, 
but  still  lacks  an  absolutely  satisfactory  solution. 

Explanations  for  the  existence  of  natural  immu- 
nity are  founded  on  the  protection  afforded:  first,  by 
the  external  and  internal  surfaces  of  the  body ;  second, 
by  inflammatory  processes ;  third,  by  natural  antibac- 
terial and  antitoxic  substances,  and  fourth,  by  the 
natural  metabolic  activity  or  vital  resistance  of  the 
organism. 

Bacteria  are  unable  to  penetrate  the  unbroken  cu- 
taneous epithelium,  but  may  reach  the  subcutaneous  tis- 
sues through  abrasions  although  microscopical  in  size, 
through  sudoriferous  and  sebaceous  ducts  and  glands 
although  their  secretions  are  mildly  antibacterial,  and 
through  the  hair  follicles.  In  the  subcutaneous  tissues, 
bacterial  encroachment  is  further  combated  by  cellular 
proliferation  and  extravasated  plasma  containing 
serum,  fibrinogen,  and  leucocytes.  Bacteria  entering 
the  nasal  and  oral  passages  encounter  in  the  mucus 
and  saliva  both  physical  and  chemical  barriers.  The 
gastric,  biliary  and  pancreatic  juices  exert  antibac- 
terial and  neutralizing  functions. 

Inflammation,  a  manifestation  of  tissue  injury,  is 


4  APPLIED  IMMUNOLOGY 

a  process  designed  to  resist  infection,  if  that  be  its 
cause,  and  proves  successful  for  mechanical  and  im- 
munological reasons,  if  the  reactive  forces  of  the  in- 
dividual be  capable.  The  invading  bacteria  develop  a 
condition  of  positive  chemotaxis  and  leucocytes 
swarm  to  the  battle-field.  Already  the  bacteria  have 
stimulated  the  tissue  cells  to  the  production  of  specific 
substances  or  antibodies  and  many  bacteria  are  killed 
by  lysis  (see  Chapter  XI)  ;  the  remainder,  by  virtue 
of  sensitization  with  their  specific  antibodies  (opsonins 
of  Chapter  XX),  are  ingested  and  destroyed  by  the 
phagocytes  (see  Chapter  XVII).  While  the  mortal 
conflict  between  bacteria  and  bacteriolysins  and  phag- 
ocytes is  being  waged,  inflammatory  exudate  and 
proliferation  of  fi:xed  tissue  cells  occur  and  raise  bar- 
riers to  the  further  extension  of  the  morbid  process. 
Coincidently,  the  antibodies  are  formed  in  excess  and 
impregnate  the  blood-serum,  establishing  the  phenom- 
enon of  immunity. 

There  is  little  evidence  pointing  to  the  presence  of 
natural  antitoxic  substances  in  animals,  although  they 
have  been  claimed  to  occur  to  a  limited  extent  in  horses 
and  more  abundantly  in  children  and  adults.  Natu- 
ral antibacterial  substances  are  more  extensively  de- 
monstrable in  the  tissue  fluids  and  blood-serum. 
Buchner  has  given  the  name  "  alexins  "  to  these  nor- 
mal bacteriolytic   substances.      They   appear   to   be 


INTRODUCTION  5 

identical  with  opsonins  and  may  be  increased  by  active 
immunization. 

Acquired  Immunity, — Acquired  immunity  is  that 
condition  of  protection  against  disease,  resulting  from 
recovery  from  infection  or  arising  by  virtue  of  arti- 
ficial inoculation.  It  may  be  produced  in  two  ways, 
namely,  by  active  or  passive  immunization. 

Active  immunization  signifies  the  process  by  which 
the  bodily  cells  of  an  animal  are  sti7nulated  by  a  toocin 
or  foreign  body  {antigen)  to  the  production  of  cer- 
tain other  bodies  {antibodies)  specific  against  the 
given  foreign  substance  (see  Chapter  II).  Thus  the 
animal  is  actively  concerned  in  the  elaboration  of  its 
own  antibodies,  hence  the  process  is  termed  active. 
Untreated  disease  terminates  in  either  one  of  three 
ways — death,  recovery,  or  chronicity.  If  the  viru- 
lence of  the  infection  is  great  and  the  dose  large  or 
overwhelming,  the  animal  succumbs,  especially  if  his 
vital  resistance  be  slight.  If  the  infection  is  relatively 
avirulent,  even  though  the  dose  be  large,  recovery  al- 
though protracted  may  take  place  provided  the  ani- 
mal's resistance  is  great.  If  the  degree  of  virulence  of 
the  infection  be  merely  the  average,  but  the  vital  resis- 
tance of  the  animal  be  only  mediocre,  its  cellular  ac- 
tivity or  infective  dose  may  prove  inadequate  for  the 
normal  generation  of  sufficient  specific  antibodies  to 
insure  immunity,  and  chronic  invalidism  results.  Thus 


6  APPLIED  IMMUNOLOGY 

in  patients  suffering  from  chronic  infections,  particu- 
larly, and  also  in  certain  acute  conditions,  the  number 
of  specific  antibodies  may  be  materially  increased  and 
convalescence  shortened  or  recovery  insured  by  artifi- 
cial inoculation  employing  homologous  bacteria.  This 
result  may  be  achieved  by  injecting  the  bacteria  with  a 
hypodermic  syringe  directly  into  the  tissues,  or  by 
carefully  regulated  and  graduated  auto-inoculation, 
by  manipulations,  as  massage,  hyperemia,  etc.,  of  the 
infected  area.  The  discovery  of  the  feasibility  of  pro- 
ducing auto-inoculation  in  patients  afflicted  with  a 
localized  infection  was  one  of  great  magnitude  and 
vital  consideration  in  the  study  and  correct  interpreta- 
tion of  infection  and  immunity,  particularly  in  connec- 
tion with  bacterin  therapy.  By  its  utilization  difficult 
diagnoses  have  been  established,  successful  treatment 
conducted  in  selected  cases  and  in  constitutional  dis- 
eases, clinical  interpretations  correctly  deduced,  and 
proper  treatment  applied  (see  Chapter  XXI) .  It  is 
evident,  therefore,  that  when  an  individual  recovers 
from  an  infectious  disease  he  enjoys  for  an  indefinite 
time  immunity  against  repeated  attacks  due  to  the 
same  infective  organism.  It  can  also  be  readily  under- 
stood that  immunity  against  particular  diseases  can  be 
conferred  by  artificial  inoculation,  using  the  specific 
bacteria,  viruses,  etc.  (prophylactic  inoculation  or 
vaccination). 


INTRODUCTION  7 

The  methods  by  which  immunity  may  be  acquired 
through  active  immunization  are  as  follows,  enu- 
merated in  their  order  of  efficiency: 

1.  Inoculation  with  virulent  living  bacteria,  typi- 
fied by  the  subcutaneous  injection  of  spirilla  of  Asiatic 
cholera. 

2.  Inoculation  with  attenuated  or  relatively  aviru- 
lent  microorganisms.  This  method  is  exemplified  by 
vaccination  against  smallpox  and  antirabic  inocula- 
tion. 

3.  Inoculation  with  dead  bacteria.  This  embraces 
the  prevalent  practice  of  bacterin  as  prophylactic 
(vaccine)  therapy,  notable  examples  of  which  include 
bacterial  inoculations  against  typhoid  fever,  plague, 
tuberculosis,  furunculosis,  carbunculosis,  etc.  A 
higher  immunity  may  be  produced  by  supplementing 
the  dead  bacterins  with  inoculations  of  attenuated  and 
finally  living  virulent  bacteria. 

4.  Inoculation  with  the  excreted  bacterial  prod- 
ucts. The  injection  of  the  horse  with  the  tetanus 
toxin  for  the  production  of  antitoxin  is  the  familiar 
example  of  this  method. 

5.  Inoculation  with  the  disintegrated  products  of 
dead  bacteria  (autolysates).  Little  of  value  has  at- 
tended this  procedure  and  for  practical  purposes  it 
may  be  disregarded. 


8  APPLIED  IMMUNOLOGY 

Passive  immunization  signifies  the  process  by 
which  immunity  is  acquired  when  artificial  antisera  are 
injected  into  the  animal  body  (see  Chapter  V) .  Thus 
the  inoculated  animal  plays  no  part  in  the  production 
of  the  antibodies  or  antitoxin  which  he  receives,  and 
the  process  is  termed  passive.  Hence  the  animal  is  in- 
jected with  the  specific  cellular  products  (antitoxin) 
of  another  animal  previously  actively  immunized,  and 
by  a  process  of  simple  neutralization  the  toxins  in  the 
diseased  animal  are  destroyed  and  immunity  con- 
ferred. In  passive  immunization  the  antibodies  bear  a 
close  chemical  combination  to  the  cells.  The  acquired 
immunity  of  passive  immunization  is  of  vastly  shorter 
duration  than  that  resulting  from  active  immunization. 

Mechanism  of  the  Production  of  Immunity 

There  is  a  group  of  foreign  chemical  substances, 
conveniently  styled  antigens,  to  which  the  animal  body 
reacts  in  a  definite  manner.  This  group  must  be  dif- 
ferentiated from  and  not  confounded  with  the  poison 
group,  as  the  common  poisons  are  not  antigens.  The 
group  embraces  the  foreign  proteins,  including  the 
bacterial  proteins,  also  certain  complex  and  more  or 
less  unknown  bodies,  as  bacterial  toxins,  parenteral 
proteolytic  ferments,  enzymes  and  other  animal  and 
vegetable  toxic  substances.  Following  the  subcutane- 
ous, intra-abdominal  or  intravenous  injection  of  an 


INTRODUCTION  9 

appropriate  quantity  of  such  an  antigen,  after  a  vari- 
able interval,  during  which  a  leucopaenia  may  exist, 
leucocytosis  supervenes.  After  an  incubation  period 
of  a  few  days  to  several  weeks,  it  is  found  that  the 
body  fluids,  particularly  the  blood-serum,  react  in  a 
novel  manner,  that  is,  they  possess  the  property  of 
neutralizing  the  antigen.  Although  these  are  known 
biological  facts,  explanations  as  to  when  and  how  these 
antibodies  are  formed  and  when  and  how  they  unite 
with  their  specific  antigens  are  pure  speculation. 
Nevertheless,  immunologists  generally  have  accepted 
the  ingenious  so-called  side-chain  theory  of  Ehrlich, 
which  conception,  although  regarded  as  too  visionary 
by  some,  has  sufficed  in  a  remarkable  manner  as  a 
working  basis  for  almost  all  the  great  problems  and 
discoveries  in  serology  (see  Chapter  III).  On  the 
other  hand,  JNIetchnikofF's  doctrine  of  phagocytosis 
(Chapter  XVII),  not  in  its  original  simplicity,  plays 
a  not  unimportant  role  in  the  mechanism  particularly 
of  active  immunization  in  view  of  the  researches  of  the 
opsonic  school.  The  efficiency  of  a  polynuclear  leuco- 
cytosis in  pneumonia  has  long  been  recognized  as  of 
momentous  prognostic  value  and  undoubtedly  is  inti- 
mately concerned  in  the  production  of  immunity. 

The  bactericidal  and  cytolytic  substances  normally 
present  in  the  blood-serum  and  body  fluids  of  some 
animals,  and  capable  of  marked  increase  by  antigenic 


10  APPLIED  IMMUNOLOGY 

inoculations,  are  extremely  important  considerations 
in  immunology  ( see  Chapter  XI ) . 

Other  notable  antibodies  conspicuous  in  serologi- 
cal work  are  agglutinins  (Chapter  IX),  important  in 
certain  bacterial  agglutination  tests,  and  precipitins 
( Chapter  X ) ,  of  value  in  the  identification  of  bloods 
in  forensic  medicine.  The  relationship  of  these  spe- 
cific antibodies  to  the  immunity  of  the  body  is  in  doubt. 

The  production  of  anaphylaxis,  hypersuscepti- 
bility  or  supersensitiveness  to  all  proteins  save  gelatin, 
and  its  association  with  immunization  contributes  a 
most  interesting  and  important  subject  and  is  fully 
discussed  in  Chapter  IV. 

History  axd  Development  of  Immunology 

Historically,  the  first  reference  to  any  attempt  at 
protection  against  disease  by  the  utilization  of  biologi- 
cal products  was  made  by  Mithradates,  who,  it  is  al- 
leged, took  small  quantities  daily  of  certain  poisons  in 
order  to  render  himself  immune.  Similarly,  the  cus- 
tom of  hunters  of  certain  wild  tribes  to  inoculate  them- 
selves systematically  with  snake  venom  to  safeguard 
the  effects  of  snake  bites  is  well  known. 

Immunization,  however,  was  not  placed  on  a  truly 
practical  basis  until  the  eighteenth  century,  although 
the  practice  had  existed  in  the  East  a  long  time  pre- 
viously.   Lady  JNIontagu,  the  wife  of  the  British  Am- 


INTRODUCTION  11 

bassador  to  Constantinople,  permitted  her  son  to  be 
inoculated  against  smallpox  with  the  matter  from 
variola  pustules  and  subsequently,  in  1721,  introduced 
the  method  in  England.  Despite  the  brilliant  results 
that  followed,  the  practice  encountered  bitter  opposi- 
tion, owing  to  the  fact  that  although  it  protected  the 
inoculated  it  did  not  prevent  conveyance  of  the  disease 
in  a  virulent  form  to  the  uninoculated,  and  was  finally 
prohibited  by  law. 

Sixty  years  later,  the  attention  of  Jenner  was  di- 
rected to  a  peculiar  disease  of  the  udders  of  cows,  from 
which  the  hands  of  milkmen  became  infected,  render- 
ing them  immune  to  smallpox.  Jenner  investigated 
the  subject  for  a  number  of  years,  and  in  1796  in- 
oculated a  boy  with  "  cow-pox,"  after  which  inocula- 
tion with  smallpox  showed  him  to  be  immune.  Two 
years  later,  in  1798,  Jenner  published  his  classical  re- 
port, which  was  soon  followed  by  systematic  and  uni- 
versal vaccination  against  the  world's  greatest  scourge. 
Although  the  causative  organism  in  the  virus  of  small- 
pox has  not  been  discovered,  even  to  this  day,  Jenner 
firmly  established  the  doctrine  that  it  is  possible  to  con- 
fer immunity  against  an  infectious  disease  by  the  em- 
ployment of  a  modified  materies  morhi. 

Almost  a  century  passed  before  any  further  not- 
able advance  occurred  in  immunology.  Indeed  it  re- 
quired the  stimulus  of  the  era  of  bacteriology  to  pro- 


n  APPLIED  IMMUNOLOGY 

mote  research  along  this  line.  Schwann  showed  the 
relationship  between  decomposition  of  organic  bodies 
and  microorganisms.  This  was  supplemented  by 
Pasteur's  work  on  fermentation  and  the  yeast  fungus. 
In  1863  Davaine  pointed  out  that  certain  bacilli  de- 
scribed in  the  blood  by  him  thirteen  years  before  were 
the  causative  factors  in  anthrax.  Thirteen  years  later, 
Koch  succeeded  in  growing  these  bacilli  on  an  artificial 
medium  in  pure  culture,  and  anthrax  was  reproduced 
in  animals. 

The  next  problem  was  to  attenuate  the  bacteria, 
that  is,  preserve  their  identity  and  life,  at  the  same 
time  reducing  their  virulence,  so  that  inoculation  would 
not  result  fatally.  In  1880,  Pasteur  succeeded  in 
preparing  a  "  vaccine "  from  attenuated  anthrax 
bacilli  and  inoculated  sheep,  thereby  rendering  them 
immune. 

In  1885,  after  much  animal  experimentation, 
Pasteur  inoculated  the  first  human  subject  against 
rabies.  The  etiological  microorganism  of  the  virus  of 
this  disease  is  still  undetermined  and  the  procedure  of 
antirabic  inoculation  has  undergone  no  material 
change. 

Thus  far  the  belief  prevailed  that  immunity  oc- 
curred only  as  the  result  of  recovery  from  disease. 
Salmon  and  Smith  at  this  time  demonstrated  that 
immunization  could  be  produced  by  the  products  of 


INTRODUCTION  IS 

bacterial  growth.  Supplementing  the  work  of  Traube 
and  Gscheidlen,  in  1887,  Buchner  showed  the  specific- 
ity of  the  bactericidal  properties  of  blood-serum. 

In  1888,  Roux  and  Yersin,  and  Kitasato  dis- 
covered the  toxins  respectively  of  diphtheria  and 
tetanus.  In  1890,  Behring  discovered  antitoxin  in 
the  serum  of  animals  immunized  against  the  toxin  of 
diphtheria,  thus  furnishing  the  first  antitoxic  serum. 
In  the  same  year,  with  the  collaboration  of  Kitasato, 
immunity  to  tetanus  was  conferred  on  mice  from  the 
serum  of  rabbits  inoculated  with  tetanus  toxin.  In 
the  following  year  with  Wernicke,  he  immunized  other 
animals  with  antidiphtheric  serum.  Behring,  there- 
fore, discovered  the  whole  principle  underlying  serum 
therapy  in  its  relationship  to  modern  therapeusis  and 
serological  studies,  and  became  thereby  its  honored 
founder. 

The  following  year  Ehrlich  demonstrated  anti- 
bodies in  the  serum  of  animals  inoculated  with  vege- 
table poisons,  as  ricin,  abrin  and  crotin,  and  three 
years  later  Calmette  claimed  similar  results  with  snake 
venom. 

Although  it  was  recognized,  even  before  Behring' s 
discovery,  that  the  serum  of  animals  inoculated  with 
certain  bacteria  possessed  a  specific  anti-infectious 
effect  or  protection,  it  was  about  this  time,  1892,  that 
Metchnikoff  called  attention  to  a  particular  substance, 


14  APPLIED  IMMUNOLOGY 

"  stimulin,"  in  antibacterial  sera,  capable  of  stimulat- 
ing leucocytes  to  increased  ingestion  and  destruction 
of  bacteria.  JNIetchnikoiF  claimed  that  immunity  de- 
pended upon  this  property  of  phagocytosis. 

It  was  at  this  time  that  Buchner  described 
"  alexins,"  and  Bordet  "  sensibilisators  "  in  sera  and 
their  role  in  the  process  of  immunization.  Numerous 
foreign  albuminous  substances  were  utilized  for  the 
production  of  antibodies  and  conceptions  respecting 
the  nature  of  immunity  definitely  distinguished  two 
types,  active  and  passive.  JMoreover,  it  was  realized 
that  eccotoocic  bacteria  were  adaptable  chiefly  to  pas- 
sive, and  endotoxic  bacteria  to  active,  immunization. 

In  1895,  Pfeiffer  demonstrated  his  phenomenon 
of  bacteriolysis,  thus  dealing  a  severe  blow  to  INIetchni- 
koiF's  doctrine.  While  the  discussion  was  still  warm 
over  PfeifFer's  discovery,  in  the  next  year  Gruber  and 
Durham  directed  attention  to  the  diagnostical  value  of 
"  agglutination  "  of  bacteria  in  their  specific  antiserum 
and  Widal  described  the  serodiagnosis  of  typhoid 
fever.  The  following  year,  Kraus  described  other 
antibodies,  more  or  less  closely  related  to  bacteriolysins 
and  agglutinins,  which  he  styled  "  precipitins." 

The  year  1896  is  notable  in  immunological  annals 
as  marking  the  advent  of  antityphoid  inoculation  by 
Wright.  Previously,  Ferran  and  Haffkine  had  em- 
ployed attenuated  living  cultures,  prophylactically. 


INTRODUCTION  15 

against  cholera.  Wright's  work,  however,  marked  a 
new  epoch,  since  he  demonstrated  that  antibodies  may 
be  produced  by  inoculation  with  dead  bacteria.  The 
following  year,  Haffkine  immunized  himself  against 
the  plague  by  inoculation  with  a  sterilized  culture  of 
B,  pestis.  Up  to  this  time,  bacterial  inoculations  on 
the  human  had  been  employed  solely  in  a  prophylactic 
capacity.  Wright  enjoys  the  distinction  of  being  the 
first  to  realize  that  any  bacterium  responsible  for  local 
disease  and  capable  of  isolation  in  pure  culture  may 
be  employed  in  the  form  of  a  bacterial  suspension  or 
bacterin  to  cure  the  disease  it  causes. 

Seven  years  later,  Wright  and  Douglas,  taking  ad- 
vantage of  Leishman's  studies  on  comparative  phag- 
ocytosis, showed  that  phagoc}i;osis  does  not  occur  save 
in  the  presence  of  serum,  thus  claiming  a  specific  sensi- 
tization of  bacteria  by  certain  substances  in  the  blood- 
serum  to  which  the  name  "  opsonins  "  was  given.  An 
ingenious  and  clever  laboratory  method  was  devised 
whereby  the  measure  of  the  ratio  of  phagocytability 
could  be  determined  and  this  was  styled  the  "  opsonic 
index."  The  work  of  Wright  attracted  universal  at- 
tention and  popularized  active  immunization  by  bac- 
terin therapy  to  an  unprecedented  and  world-wide 
extent. 

Synchronously  with  Wright's  studies,  the  discov- 
eries and  advances  in  serology  were  truly  remarkable. 


16  APPLIED  IMMUNOLOGY 

In  1898,  Belfanti  and  Carbone  discovered  that  the 
serum  of  horses  immunized  with  the  blood  of  rabbits 
was  very  toxic  for  these  animals.  The  analogy  be- 
tween these  cytotoxins  or  cytolysins  and  the  specific 
antibodies  formed  after  bacterial  inoculations  was  im- 
mediately realized.  Experiments  by  Bordet,  Ehrlich 
and  iMorgenroth  demonstrated  the  presence  of  haemol- 
ysins  in  the  serum  of  animals  inoculated  with  red 
blood-cells,  and  the  interesting  and  important  phe- 
nomenon of  haemolysis  or  solution  of  erythrocytes  was 
demonstrated. 

Almost  all  tissue  cells  were  employed  in  research 
work  for  the  production  of  their  specific  antibodies, 
and  to  these  various  heterogeneous  elements  the  name 
"  antigens  "  was  applied.  Thus,  leucotoxins,  spermo- 
toxins,  trichotoxins,  syncytiolysins,  hepatolysins,  ne- 
phrolysins  and  neurotoxins  were  produced  and  their 
influence  as  causative  factors  in  the  pathology  of  cer- 
tain diseases,  as  hepatitis,  nephritis,  etc.,  became  a 
much  mooted  subject  in  scientific  research.  In  1900, 
Uhlenhuth  appreciated  the  formation  of  specific  cyto- 
toxins for  carcinoma  and  sarcoma  in  patients,  and  em- 
ployed such  sera  therapeutically. 

The  year  1902  is  memorable  in  serology  as  mark- 
ing the  discovery  of  the  "  complement-fixation  reac- 
tion "  by  Bordet  and  Gengou.  They  demonstrated 
that  no  haemolysis  can  occur  if  the  thermolabile  ele- 


INTRODUCTION  17 

ment  of  haemolysin  be  absorbed  or  fixed  in  the  interac- 
tion between  antigen  and  antibody.  This  discovery 
proved  to  be  of  the  greatest  importance  and  practical 
value  and  underlies  many  modern  serological  clinical 
tests. 

Wassermann,  in  1906,  the  year  following  the  dis- 
covery of  the  Trepone7na  pallidum,  put  the  comple- 
ment-fixation or  deviation  reaction  of  Bordet  and 
Gengou  to  a  practical  and  successful  test  for  the  diag- 
nosis of  syphilis,  and  the  so-called  "  Wassermann  re- 
action "  was  the  result.  Subsequently  the  principles 
employed  in  Wassermann' s  technic  have  been  applied 
to  many  other  infectious  diseases,  notably  gonorrhoea. 

In  1909,  Ascoli,  employing  Traube's  stalagmome- 
ter,  annomiced  the  "  meiostagmin  reaction,"  and  de- 
monstrated the  feasibility  of  differentiating  between 
fluids  containing  mixtui'es  of  antigens  and  antibodies 
by  measui*ement  of  the  drops  contained  in  given  vol- 
umes. The  method  has  been  utilized  in  the  diagnosis 
of  typhoid  fever,  tuberculosis,  malignant  tumors,  foot- 
and-mouth  disease,  and  for  the  detection  of  many 
lipoidal  substances. 

Finally,  reference  should  be  made  to  certain  para- 
doxical reactions  that  have  been  observed  in  immu- 
nology during  recent  years.  Instead  of  the  immunity 
which  commonly  follows  the  injection  of  a  foreign 
protein  substance  into  an  animal,  occasionally,  al- 


18  APPLIED  IMMUNOLOGY 

though  immune  bodies  have  been  demonstrated  to  be 
present,  the  inoculation  of  heterogeneous  substances, 
without  apparent  reason,  produces  a  singular  effect, 
in  consequence  of  which  the  previously  treated  organ- 
ism reacts  differently  from  the  normal.  To  this  re- 
action von  Pirquet  has  given  the  name  "  allergy." 
Allergy  may  be  expressed  by  either  a  refractory  state 
of  the  organism,  namely,  immunity,  or  by  supersensi- 
tiveness,  that  is,  anaphylaxis.  Examples  of  anaphy- 
lactic reactions  are  recognized  in  "  Arthus'  phenome- 
non "  in  rabbits,  "  Theobald  Smith's  phenomenon  "  in 
guinea-pigs  and  "serum  disease  "  in  man.  Less  im- 
portant, although  more  practical  illustrations  are  com- 
monly seen  in  the  cutaneous  and  ophthalmic  tuber- 
culin reactions  of  tuberculous  individuals,  the  mallein 
reaction  in  glanders,  the  luetin  reaction  in  syphilis,  the 
intradermic  reaction  in  typhoidal  subjects,  the  sero- 
diagnosis  of  cancer,  etc. 

In  this  connection,  the  hypothesis  of  Friedberger 
that  a  number  of  pathological  processes  are  due  to  the 
occurrence  of  albumen  anaphylaxis,  and  the  coincident 
poisons  of  infections  are  referable  to  the  influence  of 
the  concurrent  anaphylotoxin,  must  receive  serious 
consideration. 


II 

ANTIGENS  AND  ANTIBODIES 

TOXINS  AND  ANTITOXINS— AGGLUTININS— PRECIPITINS— 
LYSINS—ISOCYTOLYSINS— OPSONINS— ANTIFERMENTS— 
AUTO-ANTIBODIES— ALLERGY  AND  ANAPHYLAXIS 

We  will  now  discuss  briefly  the  various  defensive 
properties  of  the  fluids  and  tissues  of  the  body  in  re- 
sisting the  invasion  of  disease-producing  substances. 
The  latter  are  usually  termed  antigens,  while  the  spe- 
cific substances  produced  by  the  body-cells  to  neu- 
tralize and  thus  render  the  antigens  innocuous  are 
termed  antibodies.  By  the  term  antigen,  therefore,  is 
meant  an  organic  foreign  substance,  usually  protein 
in  nature,  the  product  of  animal  or  vegetable  cells, 
which  on  being  introduced  into  the  body  has  the  prop- 
erty of  stimulating  the  production  by  the  body-cells  of 
a  specific  substance  or  antibody  which  unites  chemi-^jjn^^,,^^^^^^^ 
cally  with  it  and  thus  neutralizes  it.  Antigens  may  be 
of  various  types.  They  may  consist  of  the  soluble 
products  of  bacterial  growth,  of  substances  bound  up 
in  the  bodies  of  the  bacteria  themselves,  of  protein  and 
lipoid  extracts  of  animal  tissues,  etc.  These  different 
forms  of  antigens  produce  corresponding  types  of 
antibodies  which  differ  in  their  methods  of  attacking 
the  antigens.    Antibodies  thus  specifically  produced, 

19 


20  APPLIED  IMMUNOLOGY 

as  distinguished  from  those  naturally  existing  in  the 
normal  animal,  are  called  immune  bodies,  and  the 
animal  is  said  to  be  immune  to  the  particular  antigen 
in  question. 

Various  Forms  of  Antibodies 

(a)  Toxins  and  Antitoxins, — Toxins  are  the  solu- 
ble products  of  bacterial  and  plant  growth,  also  the 
secretion  of  certain  reptiles,  and  in  the  case  of  some 
bacteria  are  the  means  through  which  the  deleterious 
effects  of  the  bacteria  are  brought  about.  Antitoxins 
are  specific  substances  produced  in  the  blood-serum 
whereby  the  action  of  bacterial  toxins  is  antagonized. 

{h)  Agglutinins. — JVIany  forms  of  bacteria,  when 
introduced  into  the  body,  stimulate  the  body-cells  to 
produce  specific  substances  called  agglutinins,  which 
have  the  property  of  causing  the  bacteria  to  lose  their 
motility  and  to  mass  together  in  clumps.  This  phe- 
nomenon is  of  great  practical  importance  in  the  diag- 
nosis of  certain  bacterial  infections.  For  example, 
the  blood- serum  of  a  patient  affected  with  typhoid 
fever  contains  agglutinins,  so  that  when  it  is  brought 
in  contact  with  typhoid  bacilli  agglutination  of  the 
latter  will  take  place. 

{c)  Precipitins, — Precipitins  are  closely  allied  to 
agglutinins,  and  are  antibodies  which  bring  about  the 
precipitation  of  soluble  foreign  proteins.     They  also 


ANTIGENS  AND  ANTIBODIES  21 

have  considerable  diagnostic  importance,  to  which  at- 
tention will  be  called  later. 

(d)  Lysins, — Lysins  are  among  the  most  impor- 
tant of  all  antibodies,  both  from  the  stand-point  of 
protection  against  disease  and  also  from  the  fact  that 
many  diagnostic  tests  are  based  upon  their  action. 
Lysins  are  antibodies  that  bring  about  the  solution  of 
cells,  and  the  term  cytolysin  may  therefore  be  applied 
to  them  all.  Lysins  which  bring  about  the  solution  of 
bacteria  are  termed  bacimwlysins ;  those  which  cause 
solution  of  red  blood-cells  are  known  as  hcEinolysins, 
Thus,  the  terms  cytolysis,  bacteriolysis  and  hcemolysis 
should  be  readily  understood.  Lysins  are  formed  as 
the  result  of  introduction  of  bacteria  or  of  cells  of  an 
animal  of  an  alien  species  into  the  body.  Thus,  the 
red  blood-cells  of  the  sheep,  when  introduced  into  the 
body  of  the  rabbit,  produce  antibodies  or  h^emolysins 
in  the  rabbit's  blood-serum,  which  when  brought  sub- 
sequently in  contact  with  sheep's  corpuscles  under  cer- 
tain conditions  will  dissolve  the  latter. 

{e)  Isocytolysins. — It  has  been  found  that  lysins 
may  not  only  be  produced  against  the  cells  of  an  ani- 
mal of  a  foreign  species  (heterolysins)  but  also  that 
the  introduction  into  the  body  of  cells  of  an  animal  of 
the  same  species  will  cause  the  formation  of  specific 
cytolytic  antibodies  (isocytolysins). 

(/)   Opsonins, — Opsonins  are  substances  present 


22  APPLIED  IMMUNOLOGY 

in  the  blood-serum  which  prepare  bacteria  for  phag- 
ocytosis or  absorption  by  the  leucocytes.  Normal 
opsonins  are  present  in  ever}^  seruni.  Opsonins  which 
are  called  forth  by  the  introduction  of  particular  bac- 
teria are  called  immune  opsonins  or  bacteriotropins. 
They  are  more  resistant  to  heat  (thermostable)  than 
normal  opsonins. 

{g)  Antiferments. — These  are  antibodies  which 
resist  the  action  of  the  different  ferments.  Owing 
to  their  presence,  self-digestion  by  the  various  cells  of 
the  body  is  probably  prevented.  Thus  for  the  fer- 
ments, pepsin,  trypsin,  rennin,  lipase,  etc.,  we  have  the 
corresponding  antipepsin,  antitrypsin,  antirennin, 
antilipase,  etc. 

{h)  Aiito-antihodies. — It  has  further  been  shown 
that  an  animal  can  be  made  to  form  antibodies  against 
its  own  cells  when  these  are  introduced  parenterally. 

Allergy 

To  the  altered  condition  of  an  animal  into  whose 
tissues  has  been  introduced  an  antigen  or  foreign  cell 
product,  von  Pirquet  has  given  the  term  allergy. 
Under  allergic  phenomena  are  therefore  included  all 
subsequent  reactions  on  the  part  of  the  body  as  a  re- 
sult of  the  parenteral  introduction  of  foreign  protein 
materials,  i.e.,  introduction  by  other  channels  than 
through  the  gastro-intestinal  tract.    An  animal  thus 


ANTIGENS  AND  ANTIBODIES  23 

treated  is  said  to  be  "  sensitized,"  i.e.,  its  cells  have  pro- 
duced an  excess  of  antibodies  against  the  particular 
foreign  material  introduced. 

Anaphylaxis 

Anaphylaxis  is  the  term  given  by  Richet  to  a  state 
of  hypersusceptibility  or  supersensitiveness  to  the  ac- 
tion of  a  foreign  protein,  after  the  animal  has  been 
first  "  sensitized  "  by  an  injection  of  the  protein  in 
question.  The  phenomena  of  anaphylaxis  will  be 
discussed  in  greater  detail  in  Chapter  IV. 


Ill 

EHRLICH'S  SIDE-CHAIN  THEORY 

Our  present  view  of  the  mechanism  of  the  de- 
fensive processes  of  the  body  against  disease  is  based 
upon  the  side-chain  theory  of  Ehrlich.  This  theory- 
explains  the  interaction  of  the  various  antigens  and 
antibodies  and  is  almost  completely  supported  by  ex- 
perimental evidence.  It  is  most  important  to  realize 
that  the  interaction  between  antigens  and  antibodies  is 
of  a  chemical  nature,  i.e.,  the  antibody  does  not  destroy 
the  antigen  but  forms  a  chemical  combination  with  it. 
Ehrlich's  theory  conceives  of  each  body-cell  as  con- 
sisting of  a  central  molecular  complex  or  nucleus  upon 
which  its  life  depends  and  a  number  of  processes  or 
"  side  chains  "  capable  of  combining  with  foodstuffs 
for  the  nutrition  of  the  cell  and  with  foreign  substances 
which  might  prove  injurious  to  it.  These  side  chains 
or  processes  are  termed  receptors.  Each  receptor  has 
a  special  affinity  for  a  certain  kind  of  foodstuff  or 
toxin.  Certain  receptors  are  common  to  all  cells, 
while  others  are  foimd  only  in  special  cells.  It  is  also 
conceivable  that  some  receptors  may  not  be  normally 
present  but  are  formed  only  by  the  stimulation  of  cer- 
tain forms  of  toxins.  The  toxic  molecule  which  unites 
with  the  cell  receptor  consists  of  two  groups,  a  hapto- 

24 


■o  3 
5  3 


O  3J 

ai  to 
.3 


EHRLICH'S  SIDE-CHAIN  THEORY  25 

phore  group,  which  binds  it  to  the  cell  receptor,  and  a 
toxophore  group,  which  actually  bears  its  toxic  prop- 
erties. When  the  receptor  combines  with  the  toxin 
molecule,  the  cell  throws  it  off  into  the  circulation,  and 
similar  receptors  are  formed  to  take  its  place.  These, 
however,  are  formed  in  excess,  and  the  cell  throws 
them  off  also.  These  free  receptors  then  imite  with 
corresponding  toxin  molecules  in  the  circulation. 
There  is  a  receptor  for  each  particular  form  of  toxin 
molecule.  Thus  the  diphtheria  toxin  combines  only 
with  the  specific  receptor  provided  for  it,  and  will  not 
unite  with  those  intended  for  tetanus  toxin.  Ehrlich's 
theory,  which  at  first  only  covered  the  simple  union  of 
toxin  with  antitoxin,  was  extended  also  to  explain  the 
action  of  more  complex  antibodies,  and  may  be  said  to 
cover  completely  all  forms  of  antigen-antibody  ac- 
tion. There  are  three  recognized  types  or  orders  of 
receptors,  which  are  conveniently  illustrated  by  the 
accompanying  diagram  (Fig.  1).  Receptors  of  the 
first  order  possess  only  a  single  haptophore  or  com- 
bining group,  by  which  they  unite  with  the  hapto- 
phore group  of  the  corresponding  antigens.  To  this 
order  belong  antitoxins  and  antiferments.  Receptors 
of  the  second  order,  in  addition  to  the  haptophore 
group,  possess  a  second  or  zymophore  group,  by 
means  of  which  the  anchored  antigen  can  be  subjected 
to  further  change.     Under  this  head  belong  agglu- 


26  APPLIED  IMMUNOLOGY 

tinins  and  precipitins.  Receptors  of  the  third  order 
possess  two  combining  groups,  and  are  termed  ambo- 
ceptors. The  first  of  these  anchors  the  antigen  to  the 
cell  and  is  known  as  the  cytophilic  group,  while  the 
second  combines  with  the  complement  of  the  blood- 
serum  and  is  known  as  the  complementophilic  group. 
Thus,  for  the  action  of  receptors  of  the  third  order,  an 
outside  substance,  the  complement  of  the  blood-serum, 
is  necessary.  Among  these  are  cytolysins  (bacterioly- 
sins,  h^emolysins,  opsonins,  etc.).  It  is  mmecessary 
to  go  into  the  experimental  evidence  that  has  been 
brought  forward  in  support  of  Ehrlich's  theoiy  of  im- 
munity, but  numerous  investigators  have  fully  con- 
firmed his  views  by  experiments. 

It  is  well  known  that  poisons  of  known  composi- 
tion, such  as  alkaloids,  glucosides,  etc.,  when  intro- 
duced into  the  body  do  not  produce  antibodies,  thus 
differing  essentially  from  true  antigens.  The  true 
antigens  are  closely  related  to  foodstuffs  and  conse- 
quently have  an  intimate  chemical  relation  with  the 
body-cell,  which  provides  special  receptors  for  them. 
The  poisons,  on  the  other  hand,  not  having  this  spe- 
cial chemical  affinity,  are  not  closely  bound  to  the  cell- 
substance,  but  become  physically  stored  up.  Many 
of  these  substances  can  be  recovered  from  the  body  by 
extraction,  which  could  not  occur  if  chemical  union 
had  taken  place. 


IV 

ANAPHYLAXIS  OR  HYPERSUSCEPTIBILITY 
Ix  connection  with  the  administration  of  antisera 
for  therapeutic  purposes,  the  possibihty  of  the  occur- 
rence of  the  phenomenon  known  as  anaphylactic  shock 
must  be  borne  in  mind. 

The  phenomenon  of  protein  sensitization  was  rec- 
ognized by  Vaughan,  von  Behring  and  others  for 
many  years  before  the  name  anaphylaaJis,  under  which 
it  is  now  generally  known,  was  given  by  Richet  in 
1911.  The  term  indicates  absence  of  protection,  as 
opposed  to  prophylaxis.  Anaphylaxis  consists  in  a 
series  of  apparently  deleterious  effects  produced  by  a 
second  injection  of  specific  protein  material  into  an 
animal  that  has  been  previously  *'  sensitized  "  by  a 
first  injection  of  the  same  material.  For  instance,  if 
an  animal  receive  one  injection  of  a  foreign  protein 
material,  say  of  antitoxic  serum,  no  deleterious  effects 
are  usually  observed,  but  if  this  injection  be  repeated 
after  several  days,  even  in  a  very  small  dose,  the 
second  injection  may  be  followed  immediately  by  a 
marked  reaction,  such  as  convulsions  and  respiratory 
difficulty,  in  some  cases  with  a  fatal  result.  The  ana- 
phylactic phenomena  usually  follow  when  the  second 
injection  is  given  at  least  twelve  days  after  the  first. 

27 


28  APPLIED  IMMUNOLOGY 

One  of  the  earliest  investigators  to  devote  special  at- 
tention to  this  subject  was  Richet.  He  injected  an 
animal  with  a  dose  of  protein  poison,  and  after  com- 
plete recovery  from  the  symptoms  produced,  by  inject- 
ing a  very  much  smaller  dose  which  in  a  non-sensitized 
animal  would  have  no  deleterious  effects,  was  able  to 
cause  death  in  a  few  hours.  This  hypersusceptibility 
in  the  sensitized  animal  Richet  ascribed  to  the  forma- 
tion of  a  special  antibody  by  the  first  injection,  which 
on  the  second  injection  causes  the  splitting  off  of  a 
highly  toxic  substance  from  the  toxin  injected,  giving 
rise  to  the  symptoms.  Richet's  conclusions  were  based 
on  the  assumption  that  the  phenomena  only  occurred 
when  toxic  substances  were  injected.  Arthus  in  1903 
showed  that  hypersusceptibility  occurred  when  non- 
toxic protein  material  was  used,  such  as  normal  horse 
serum,  milk,  etc.  (Arthus'  phenomenon).  He  also 
showed  that  the  symptoms  could  only  be  produced  by 
means  of  the  same  substance  that  w^as  originally  in- 
jected; e.g. J  that  an  animal  first  sensitized  with  horse 
serum  would  not  be  affected  with  a  subsequent  in- 
jection of  milk  or  other  protein  material,  but  only 
with  horse  serum.  The  studies  of  von  Pirquet  showed 
that  a  certain  interval  must  elapse  after  the  first  in- 
jection of  serum  before  a  second  injection  will  pro- 
duce s}Tnptoms,  and  he  concluded  that  this  interval 
represented  a  period  of  incubation  necessary  for  anti- 


ANAPHYLAXIS  OR  HYPERSUSCEPTIBILITY      29 

body  formation.  He  applied  this  theory  to  the  period 
of  incubation  and  symptoms  of  infectious  diseases,  all 
of  which  may  be  regarded  as  anaphylactic  phenomena. 
In  short,  according  to  von  Pirquet,  the  causative 
agents  of  disease  do  not  show  their  deleterious  effects 
until  antibodies  are  formed  which  split  them  up,  set- 
ting free  toxic  substances.  Theobald  Smith  observed 
that  guinea-pigs,  which  had  been  injected  with  toxin- 
antitoxin  mixture,  were  hypersensitive  to  subsequent 
injections  with  normal  horse  serum  and  rapidly  suc- 
cumbed to  small  doses  of  the  latter.  The  most  attrac- 
tive and  rational  explanation  of  the  phenomena  fol- 
lowing protein  sensitization  or  anaphylaxis  was  first 
brought  forward  by  Vaughan  in  1907  {Jour.  Infec- 
tions Diseases,  1907,  iv,  476).  Vaughan's  views  may 
be  briefly  stated  as  follows:  Every  protein  molecule 
consists  of  a  poisonous  group  and  a  non-poisonous  or 
specific  group.  The  latter  group,  when  the  molecule 
is  introduced  into  the  body,  induces  the  development 
in  the  body  of  a  specific  proteolytic  ferment,  which 
has  the  power  of  splitting  up  similar  protein  mole- 
cules, setting  free  the  poisonous  or  non-specific  group. 
The  poison  thus  set  free  produces  the  phenomena  of 
anaphylaxis.  Upon  the  first  injection  of  a  serum, 
therefore,  the  specific  group  of  the  molecule  causes  the 
formation  in  the  body  of  a  specific  proteolytic  ferment. 
The  animal  is  now  said  to  be  *'  sensitized."    If  a  second 


30  APPLIED  IMMUNOLOGY 

dose  of  the  same  serum  then  be  given,  its  molecule  is 
split  up  by  the  proteolytic  ferment,  and  the  toxic  con- 
stituent is  set  free,  giving  rise  to  anaphylaxis.  There 
is  no  setting  free  of  the  poisonous  elements  of  the  pro- 
tein molecule  immediately  after  the  first  injection  of 
serum,  because  the  specific  proteolytic  ferment  de- 
velops gradually  in  response  to  the  stimulation  of  the 
specific  group  of  the  molecule.  Hence  the  toxic  ele- 
ment is  only  liberated  in  small  amounts  as  the  ferment 
is  formed.  But  by  the  time  a  second  injection  of 
serum  is  administered,  a  considerable  amount  of  the 
proteolytic  ferment  is  stored  up,  and  is  ready  to  split 
up  the  molecules  of  the  second  injection  and  set  free 
the  toxin  which  gives  rise  to  symptoms  of  anaphylaxis. 
The  anaphylactic  poison  affects  chiefly  the  nervous 
system,  and  particularly  the  respiratory  centres.  The 
reaction  manifests  itself  in  respiratory  difiiculty  and 
convulsions,  which  may  result  fatally,  and  is  particu- 
larly liable  to  occur  in  asthmatic  persons.  The  injec- 
tion of  antitoxic  horse  serum  has  been  known  to  pro- 
duce anaphylactic  phenomena  in  persons  in  whom  at- 
tacks of  asthma  are  induced  by  contact  with  horses. 
We  do  not  yet  know  the  nature  of  the  anaphylactic 
poison.  It  is  now  recognized  that  not  only  serum,  but 
all  forms  of  toxic  and  non-toxic  protein  material,  in- 
cluding milk,  extracts  of  normal  and  pathologic  ani- 


ANAPHYLAXIS  OR  HYPERSUSCEPTIBILITY     31 

mal  tissue,  and  bacteria,  may  give  rise  to  anaphylactic 
phenomena. 

Anti-anaphylaxis. — If  an  animal  receive  a  second 
injection  of  foreign  protein  material  before  the  twelfth 
day  after  the  first  injection,  a  condition  of  resistance 
or  decreased  susceptibility  may  be  set  up  (anti-ana- 
phylaxis), and  the  symptoms  of  anaphylaxis  will  not 
occur.  This  resistance  may  be  kept  up  by  repeated 
injections  at  short  intervals.  This  fact  is  of  impor- 
tance in  the  repetition  of  antitoxic  serum  for  therapeu- 
tic purposes.  The  mechanism  of  anti-anaphylaxis  is 
as  yet  unknown. 

Passive  Anapliylaccis, — Anaphylaxis  may  be 
brought  about  passively  as  well  as  actively.  Thus,  if 
one  animal  be  sensitized  by  an  injection  of  foreign 
protein  and  sufficient  time  be  allowed  to  elapse  for  the 
formation  of  the  specific  protein-splitting  ferments, 
introduction  of  its  serum  into  a  second  animal  will 
sensitize  the  second  animal  to  the  protein  in  question. 
Anaphylaxis  will  then  follow  immediately  after  the 
injection  of  the  second  animal  with  the  protein. 

The  anaphylaxis  reaction  is  made  use  of  in  the 
diagnosis  of  several  diseases.  The  various  tuberculin 
reactions  of  von  Pirquet,  WolfF-Eisner,  Calmette, 
]\Ioro  and  others  depend  on  this  hypersusceptibility. 
Tuberculous  persons  being  "  sensitized  "  by  the  tuber- 
culous process,  the  application  of  tuberculin  in  these 


32  APPLIED  IMMUNOLOGY 

cases  will  give  rise  to  the  various  reactions.  A  similar 
reaction  of  diagnostic  value  occurs  in  gonococcic  in- 
fection, a  rise  of  temperature  and  other  anaphylactic 
symptoms  following  the  injection  of  gonococcus  bac- 
terin  in  persons  suffering  from  this  infection. 
Noguchi's  luetin  reaction  in  syphilis  also  is  an  anaphy- 
lactic manifestation. 

Many  of  the  phenomena  of  disease  may  be  ex- 
plained on  the  basis  of  anaphylactic  or  allergic  phe- 
nomena. As  pointed  out  by  von  Pirquet,  the  incuba- 
tion period  of  an  infectious  disease  is  the  time  required 
by  the  body  to  form  specific  antibodies  to  the  infecting 
organism.  The  onset  of  symptoms  marks  the  setting 
free  of  the  toxin,  when  the  infecting  organism  is  split 
up  by  the  specific  antibody.  Studies  in  anaphylaxis 
have  also  furnished  a  very  plausible  explanation  for 
certain  individual  idiosyncrasies.  It  is  probable  that 
hay  fever  and  allied  conditions  are  manifestations  of 
anaphylaxis.  The  persons  susceptible  to  these  idio- 
sjmcrasies  are  probably  sensitized  by  the  pollen  of 
plants  and  other  substances,  so  that  they  manifest 
symptoms  w^hen  placed  in  contact  with  small  quanti- 
ties of  this  proteid.  In  the  same  way  may  be  explained 
idiosyncrasies  to  various  articles  of  food,  such  as  straw- 
berries, pork,  lobster,  exposure  to  exhalation  from 
horses,  etc. 

Certain  precautions  may  be  taken  to  avoid  ana- 


ANAPHYLAXIS  OR  HYPERSUSCEPTIBILITY     3S 

phylactic  shock  in  the  administration  of  serum  for 
therapeutic  purposes.  Rosenau  and  Anderson  point 
out  that  anaphylaxis  is  to  be  feared  in  all  persons  who 
have  shown  a  tendency  to  astlima,  or  who  have  received 
previous  injections  of  serum  at  least  twelve  days  be- 
fore. In  these  persons  it  is  well  to  make  a  preliminary 
injection  of  a  small  trial  dose  of  0.1  to  0.2  c.c.  of  serum, 
and  if  no  symptoms  appear  within  two  hours,  the  full 
dose  may  be  given. 

Serum  Sickness. — The  symptoms,  grouped  under 
the  term  "  serimi  sickness,"  occasionally  seen  after  the 
injection  of  antitoxic  serum  in  diphtheria  and  other 
diseases,  are  for  the  most  part  manifestations  of  ana- 
phylaxis. These  symptoms  usually  consist  in  fever, 
urticarial  eruptions,  lymphatic  nodal  enlargements, 
and  kidney  irritation.  Usually  these  symptoms  are 
transitory,  but  fatal  cases  have  been  reported.  In 
order  for  these  phenomena  to  follow  a  single  injection, 
as  occasionally  happens,  a  large  dose  of  serum  must  be 
employed;  but  the  appearance  of  symptoms  may  occur 
after  a  very  small  second  injection.  The  mild  cases  of 
serum  sickness  require  no  special  treatment.  With  the 
refinement  of  preparation  and  concentration  in  small 
bulk  of  antisera,  untoward  symptoms  following  in- 
jection have  become  comparatively  rare. 


ANTISERA 

PREPARATION   OF   ANTISERA— INDICATIONS   FOR  THERA- 
PEUTIC USE  OF  ANTISERA 

Therapeutic  antisera  for  protective  and  cui^ative 
passive  immunization  fall  into  two  general  classes: 
(a)  antitoxic  sera,  and  (b)  antibacterial  sera,  accord- 
ing to  the  mode  of  action  of  the  bacteria  against  which 
they  are  directed. 

Antitoxic  sera  are  sera  which  are  applied  to  the 
group  of  bacteria  whose  deleterious  action  depends 
upon  the  toxins  separated  from  them  in  the  process  of 
growth.  The  best  examples  of  this  class  of  bacteria 
are  the  bacillus  of  diphtheria  and  the  bacillus  of 
tetanus. 

Antibacterial  sera  are  employed  particularly  in  in- 
fections by  those  bacteria  whose  toxins  are  contained 
within  the  bacterial  protoplasm  (endotoxins).  They 
act  by  destroying  the  bacteria  themselves.  Generally 
speaking,  passive  immunization  has  not  been  so  suc- 
cessful in  dealing  with  infections  by  bacteria  belong- 
ing to  this  group,  as  with  those  whose  action  depends 
on  separated  or  extracellular  toxins. 

34 


ANTISERA.  35 

Preparation  of  Antitoxic  Sera 
When  bacteria  whose  action  depends  upon  toxins 
separated  from  them  in  the  process  of  growth  enter 
the  body,  they,  as  a  rule,  remain  at  the  point  of  en- 
trance, from  which  the  toxins  spread  through  the  cir- 
culation. In  response  to  the  stimulation  of  the  toxins, 
the  body  produces  antitoxins  in  the  blood-serum,  by 
which  their  deleterious  action  is  neutralized.  The  anti- 
toxins at  the  same  time  render  harmless  to  some  extent 
the  living  bacteria  at  the  point  of  entrance.  This  proc- 
ess is  termed  active  immunization. 

Antitoxin  production  is  artificially  induced  by  ac- 
tive immunization  of  suitable  animals  by  subcutaneous, 
intraperitoneal  or  intravenous  injections  of  the  toxins 
of  certain  bacteria.  The  antitoxin  thus  formed  not 
only  protects  the  animal  inoculated,  but  can  also  be 
removed  from  the  original  animal  and  used  as  a  cura- 
tive and  protective  agent  against  the  same  infection  in 
other  animals,  by  means  of  injections.  The  process  in 
the  second  animal  is  known  as  passive  immunization. 
So  we  distinguish  active  from  passive  immunization  by 
the  fact  that  in  the  former  the  animal  produces  its  own 
antibodies  to  combat  an  infection,  while  in  the  latter 
the  antibodies  have  been  produced  by  another  animal 
( see  Chapter  I ) . 

As  an  example  of  the  preparation  of  antitoxic  sera, 
the  method  of  artificial  production  of  antidiphtheritic 
serum  (diphtheria  antitoxin)  will  be  described. 


36  .  APPLIED  IMMUNOLOGY 

First  of  all  it  is  necessary  to  obtain  a  quantity  of 
the  toxin,  separated  from  the  bacteria.  The  diph- 
theria bacilli  are  grown  in  bouillon,  and  the  fluid  con- 
taining the  toxin  freed  from  bacteria  by  passage 
through  a  Berkefeld  filter.  The  toxin  strength  of  the 
fluid  must  then  be  measured.  The  unit  of  toxin 
strength  is  the  smallest  quantity  necessary  to  kill  a 
guinea-pig  weighing  250  grammes  in  5  days  or  less. 
This  quantity  of  toxin  is  called  the  minimal  lethal  dose. 
It  should  be  noted  that  not  all  strains  of  diphtheria 
bacilli  form  toxin  suitable  for  the  production  of  anti- 
toxin. For  the  formation  of  antitoxic  serum,  the 
horse  has  been  found  to  be  the  most  suitable  animal, 
but  individual  horses  difl*er  in  the  quantity  and  quality 
of  antitoxin  they  are  able  to  produce.  No  definite 
rules  can  be  laid  down  for  the  active  immunization  of 
the  horse  against  diphtheria  toxin.  But  in  a  general 
way,  small  doses  of  the  toxin  are  first  injected,  and 
gradually  increased,  until  a  very  large  quantity  of  the 
toxin  can  be  injected  at  one  time.  In  regulating  the 
amount  of  toxin  to  be  injected,  one  is  guided  by  the 
effects  of  the  previous  dose,  which  are  not  shown  im- 
mediately as  a  rule,  but  appear  after  a  few  days. 

From  time  to  time,  small  quantities  of  blood  are  re- 
moved from  the  jugular  vein  of  the  horse,  and  tested 
for  antitoxic  strength.  When  a  sufficiently  powerful 
antitoxic  content  has  been  demonstrated  in  the  horse's 


ANTISERA  37 

blood-serum,  as  described  below,  a  large  amount  of 
blood  is  drawn  off,  and  prepared  for  therapeutic  pur- 
poses. The  same  horse  can  be  used  over  and  over 
affain  for  the  formation  of  antitoxin,  if  allowed  a 
sufficient  period  for  recuperation"  after  each  bleeding. 
The  formation  of  a  sufficiently  potent  antitoxic  serum 
usually  requires  from  six  weeks  to  two  months. 

The  strength  of  antitoxin  is  measured  in  units. 
The  antitoxic  unit  is  that  quantity  of  horse's  serum 
which  will  render  harmless  the  injection  of  100  mini- 
mal fatal  doses  of  toxin.  It  is  now  ascertained  what 
is  the  smallest  quantity  of  toxin  which  when  mixed 
and  injected  with  1  antitoxin  unit  will  kill  a  guinea-pig 
of  250  grammes  in  4  or  5  days.  This  quantity  of  toxin 
is  then  mixed  with  diif erent  dilutions  of  the  horse's 
serimi  and  injected  subcutaneously  into  several 
guinea-pigs  weighing  250  grammes  each.  Supposing 
that  1/1000  c.c.  of  the  antitoxin  neutralizes  the  dose 
of  toxin  so  that  the  guinea-pig  injected  with  this  quan- 
tity remains  alive,  then  1  c.c.  of  the  horse's  serum  is 
said  to  contain  1000  units  of  antitoxin.  The  strength 
of  the  antitoxic  serum  having  been  ascertained  in  this 
manner,  it  is  placed  in  suitable  quantities  in  syringes 
for  therapeutic  use.  All  these  procedures  are  of 
course  carried  out  under  strict  aseptic  conditions. 
Antidiphtheric  serum,  like  other  antisera,  loses  its 
potency  after  variable  lengths  of  time. 


38  APPLIED  IMMUNOLOGY 

Antitetanic  and  other  antitoxic  sera  are  produced 
and  tested  in  a  similar  manner,  the  horse  being  the  ani- 
mal usually  immunized  for  their  production. 

Preparation  of  Antibacterial  Sera 

The  bacteria  belonging  to  this  group  do  not  act  by 
means  of  extracellular  toxins.  Their  deleterious  ef- 
fects depend  upon  toxins  set  free  from  the  bacterial 
protoplasm  when  the  organisms  undergo  disintegra- 
tion (endotoxins).  These  toxins  cannot  be  separated 
from  the  bacteria  themselves  in  vitro,  except  to  a  very 
slight  extent.  At  any  rate  it  is  doubtful  if  the  sub- 
stances which  call  forth  the  production  of  antibodies 
can  be  thus  separated.  Antibacterial  sera  are  there- 
fore produced  by  injecting  the  bacteria  themselves 
into  the  body  of  the  animal  that  is  to  be  actively  im- 
munized. These  are  injected,  usually  at  first  inac- 
tivated, but  also  at  times  in  an  attenuated  or  even  in 
their  living  state  (Fig.  2),  in  gradually  increasing 
doses  until  an  antibacterial  serum  of  suitable  strength 
is  obtained,  when  the  animal  is  bled,  and  the  serum 
preserved  in  ampoules  containing  convenient  doses  for 
therapeutic  injection.  The  antibodies  contained  in 
antibacterial  sera  are  not  of  the  comparatively  simple 
nature  of  those  found  in  antitoxic  sera,  but  belong  to 
the  second  and  third  orders  of  Ehrlich.  The  action  of 
antibacterial  sera  depends  upon  several  factors,  in- 


ANTISERA. 


39 


eluding  opsonins,  bacteriolysins,  and  to  some  extent 
agglutinins  and  precipitins  ( see  Chapters  IX,  X,  XI 
and  XX). 

The  standardization  of  dosage  of  antibacterial  sera 
is  therefore  more  difficult  and  uncertain  than  that  of 


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Fig.  2. — Illustrative  of  opsonic  curve,  showing  immunity  of  rabbit  inoculated  with  liv- 
ing tubercle  bacilli  of  low  virulence.    ©  =  opsonic  indices  ;    X  =  inoculations. 

antitoxic  sera,  but  the  potency  of  an  antibacterial 
serum  can  to  some  extent  be  gauged  by  determination 
of  the  opsonic  index  from  time  to  time  during  the  im- 
munization of  the  animal  (Fig.  3) ,  and  also  by  testing 
the  agglutinating  power  of  the  serum,  though  the  lat- 
ter method  is  of  only  slight  value  as  an  indication  of 


40 


APPLIED  IMMUNOLOGY 


therapeutic  strength.  The  dosage  of  antibacterial  sera 
is  chiefly  determined  by  the  therapeutic  effects  ob- 
tained. In  general,  antibacterial  sera  are  not  of  so 
much  value  as  antitoxic  sera,  though  in  the  case  of  a 


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nity of  ram.     ©  =  opsonic  indices;    X  =  inoculations  of  Staphylococcus  aureus. 

few  organisms  good  results  have  been  obtained.  Active 
immunization  offers  a  greater  field  of  usefulness  than 
passive  immunization  in  infections  of  this  type. 
Indications  for  the  Therapeutic  Use  of  Antisera 
In  the  case  of  infections  with  bacteria  which  act 
through  extracellular  toxins,  antitoxic  sera  have  a 


ANTISERA  41 

high  therapeutic  value  both  for  protective  and  cura- 
tive purposes.  By  their  use  in  persons  liable  to  be  ex- 
posed to  an  infection,  protection  for  a  limited  period 
of  time  may  be  conferred.  This  protection  by  passive 
immunization  is  not  so  durable  as  by  active  immuniza- 
tion, though  the  time  necessary  to  acquire  it  is  much 
less,  a  fact  of  considerable  importance  in  some  cases. 
It  has  been  estimated,  experimentally  and  clinically, 
that  the  duration  of  passive  immunity  persists  from 
three  to  six  weeks,  while  active  immunization  may 
endure  for  a  year  or  two.  Moreover,  studies  in  pas- 
sive immunization  have  sho^vn  that  the  duration  of 
immunity  from  homologous  antisera  lasts  three  to  four 
times  as  long  as  that  from  heterologous  immune  sera. 

Antitoxic  serum  in  some  infections  has  a  high 
curative  value  in  persons  already  suffering  from  the 
disease,  and  indeed  in  certain  infections  is  the  most 
important  means  of  treatment.  In  the  case  of  other 
diseases,  the  value  of  the  antitoxic  serum  as  a  curative 
agent  is  problematical. 

Antibacterial  sera,  in  infections  where  they  are  of 
any  value  at  all,  are  indicated  especially  to  overcome 
the  immediate  infection,  owing  to  their  rapid  effects, 
while  more  lasting  protection  in  this  class  of  infections 
is  conferred  by  active  immunization  which  pursues  a 
slower  course.  The  indications  for  active  and  passive 
immunization  in  specific  cases  will  be  taken  up  later. 


VI 

ANTITOXIC  SERA 

Antitoxic  sera  are  of  recognized  value  particu- 
larly in  diphtheria  and  tetanus,  and  are  employed  as 
routine  therapeutic  agents  in  these  diseases. 

Antitoxic  sera  have  also  been  prepared  and  used 
from  time  to  time  in  other  infections  with  varying 
success.  Examples  of  these  are  antigonococcic  serum, 
antityphoid  serum,  antituberculosis  serum,  antidysen- 
teric  serum,  anticholera  serum,  antibotulism  serum. 
In  addition,  antitoxic  sera  have  been  employed  in  hay 
fever  ( antiphy totoxic  sera),  and  against  snake  ven- 
oms (antizootoxic  sera). 

Antidiphtheeitic  Serum  (Diphtheria  Antitoxin) 

With  antidiphtheritic  serum  are  obtained  the  most 
brilliant  results  in  the  domain  of  serum  therapy.  Sta- 
tistics from  all  sources  attest  its  value.  The  serum  is 
employed  both  for  protective  and  curative  pm'poses. 
The  method  of  preparation  of  antidiphtheritic  serum 
has  already  been  given  in  Chapter  V. 

Protective  Use  of  Diphtheria  Antitoxin, — The 
average  duration  of  immunity  conferred  by  a  protec- 
tive dose  of  diphtheria  antitoxin  is  three  weeks.  This 
varies  with  the  severity  of  the  epidemic  present.      In 

42 


ANTITOXIC  SERA  43 

ordinary  epidemics  a  suitable  protective  dose  is  500 
units,  injected  subcutaneously. 

Site  and  Method  of  Injection, — The  usual  sites 
of  injection  are  under  the  skin  of  the  abdomen,  the 
thigh,  or  between  the  shoulder  blades.  The  skin  is 
thoroughly  cleansed  with  alcohol,  lifted  w4th  the 
thumb  and  forefinger,  and  the  needle  of  the  syringe 
quickly  introduced  beneath  it.  The  serum  is  allowed 
to  enter  slowly,  the  needle  quickly  withdra^^n,  the 
pmicture  site  compressed  and  stroked  with  a  pledget 
of  cotton  soaked  in  alcohol,  and  the  skin  gently 
massaged.  It  is  usually  unnecessary  to  apply  any 
dressing  at  the  site  of  puncture  of  the  skin.  Infec- 
tion rarely  follows  if  asepsis  has  been  observed. 
A  protective  dose  of  antitoxin  should  be  given  to  all 
persons  coming  in  close  contact  with  a  case  of  diph- 
theria, especially  to  children.  Some  hospitals  require 
that  all  children  admitted  receive  a  prophylactic 
injection. 

Von  Behring's  Method  of  Prophylaocis, — Recently 
von  Behring  has  brought  forward  a  new  method  of 
prophylaxis  against  diphtheria,  by  combined  active 
and  passive  immunization.  He  injects  a  mixture  con- 
sisting of  strong  diphtheria  toxin  and  antitoxin  in  such 
proportions  that  the  toxin  is  slightly  in  excess  of  the 
antitoxin.  Experimental  evidence  proves  that  more 
lasting  immunity  is  conferred  by  this  means  than  by 


44  APPLIED  IMMUNOLOGY 

the  injection  of  antitoxin  alone.  Statistics  showing 
the  increased  incidence  of  cases  of  diphtheria  in  large 
cities  demonstrate  the  necessity  for  improvement  in 
methods  of  prevention,  and  it  is  hoped  that  this 
method  will  fulfil  its  promise.  Few  statistics  are 
available  as  yet  upon  which  to  base  conclusions. 

Curative  Treatment  of  Diphtheria, — Antitoxin 
should  be  used  as  early  in  the  disease  as  possible,  for 
with  every  day  of  delay  there  is  a  great  increase  in  the 
mortality  rate.  The  death-rate  is  practically  nil  in 
patients  who  receive  injections  on  the  first  day  of  the 
disease.  The  average  dose  in  the  ordinary  case  when 
seen  on  the  first  day  is  5000  miits,  regardless  of  the  age 
of  the  patient  unless  extremely  young.  In  a  severe 
case  the  dose  should  be  10,000  or  15,000  units.  In  cases 
seen  later  the  dose  should  be  proportionately  larger, 
sometimes  as  much  as  100,000  units  being  given. 
A  single  large  dose  of  the  serum  is  preferable  to  re- 
peated smaller  doses.  Beneficial  results  should  be 
seen  from  the  injection  in  about  eight  hours,  charac- 
terized by  a  subsidence  of  the  throat  symptoms  and 
fall  of  the  temperature  and  pulse-rate.  Otherwise 
the  injection  may  be  repeated  in  larger  dose.  While 
the  administration  of  antitoxic  serum  is  by  far  the 
most  important  point  in  the  treatment  of  diphtheria, 
other  measures,  such  as  rest  in  bed,  liquid  diet,  sprays. 


ANTITOXIC  SERA  45 

and   cardiac    and    renal    therapeusis,    must    not    be 
omitted. 

Diphtheria  Carriers, — After  disappearance  of  the 
clinical  symptoms  of  diphtheria  it  is  found  that  many 
cases  still  harbor  the  bacilli  in  the  nose  and  throat  for 
variable  lengths  of  time,  thus  becoming  sources  where- 
by the  disease  is  spread  to  others.  Moreover,  many 
persons  harbor  the  organisms  who  have  never  had  an 
attack  of  the  disease.  Schiotz,  of  Copenhagen,  in 
1909,  after  noticing  that  persons  with  staphylococcic 
infections  of  the  nose  and  throat  seldom  contracted 
diphtheria,  began  to  use,  with  good  results,  a  spray 
made  from  a  culture  of  Staphylococcus  aureus  in  per- 
sons infected  with  diphtheria  bacilli.  Reports  by 
many  observers  show  almost  uniformly  good  results 
from  this  treatment.  Lorenz  and  Ravenel  {Jour,  A. 
M.  A,,  August  31,  1912,  p.  690)  recommend  a  fresh 
suspension  of  Staphylococcus  pyogenes  aureus  in  nor- 
mal saline  solution  or  a  bouillon  culture  twelve  hours 
old.  This  is  used  as  a  spray  for  the  fauces,  pharynx 
and  nose,  after  the  local  signs  of  diphtheria  have  dis- 
appeared, while  the  cultures  for  the  bacilli  are  still 
positive.  The  spray  is  repeated  at  four-hour  inter- 
vals on  two  succeeding  days,  and  continued  there- 
after until  the  cultures  become  negative  to  diphtheria 
bacilli.  Untoward  results  are  rarely  seen.  The  period 
of  quarantine  after  diphtheria  is  greatly  shortened  by 


46  APPLIED  IMMUNOLOGY 

this  method  when  compared  with  cases  treated  by  anti- 
septic sprays  alone,  and  many  cases  of  "  carriers  " 
that  have  resisted  all  other  forms  of  treatment  have 
responded  rapidly  to  it. 

Antitetanic  Serum 

Tetanus,  like  diphtheria,  is  a  disease,  manifesta- 
tions of  which  depend  upon  a  toxin  separated  from 
the  tetanus  bacilli  in  the  process  of  growth.  When 
a  wound  becomes  infected  with  tetanus  bacilli,  the  lat- 
ter remain  at  the  point  of  entrance  and  give  off  the 
toxin  which  is  disseminated  along  the  nerve  sheaths 
mitil  it  reaches  the  spinal  cord.  Here  the  toxin  com- 
bines with  the  cells  of  the  central  nervous  system,  and 
gives  rise  to  the  typical  s}Tnptoms  of  the  disease.  This 
affinity  of  tetanus  toxin  for  nerve  tissue  has  an  im- 
portant bearing  in  the  treatment  of  tetanus. 

The  method  of  artificially  producing  antitetanic 
serimi  is  similar  to  that  of  producing  antidiphtheric 
serum.  Tetanus  toxin  is  formed  by  growing  tetanus 
bacilli  anaerobically  in  bouillon  for  several  dsLjs,  the 
bacilli  being  then  removed  by  passing  the  fluid  through 
a  Berkefeld  filter.  The  filtrate  is  now  injected  into  a 
horse,  at  first  in  small  amounts  (0.5  c.c.)  mixed  with 
a  quantity  of  antitetanic  serum.  The  doses  are  then 
gradually  increased,  the  antitoxin  being  omitted  after 
the  first  few  injections.    There  are  several  methods  of 


ANTITOXIC  SERA  47 

standardization  of  tetanus  antitoxin.  The  unit  for  the 
United  States  is  the  smallest  amount  of  immunized 
horse's  serum  that  will  protect  a  guinea-pig  weighing 
350  grammes  against  1000  times  the  fatal  dose  of 
tetanus  toxin.  The  serum  having  been  standardized, 
it  is  stored  in  syringes  convenient  for  therapeutic  use. 
As  in  the  case  of  diphtheria  antitoxin,  antitetanic 
serum  loses  its  potency  after  a  variable  length  of  time. 

Therapeutic  Use  of  Antitetanic  Serum. — ^The 
therapeutic  efficiency  of  antitetanic  serum  depends 
chiefly  on  whether  it  is  able  to  reach  and  neutralize  the 
toxin  before  the  latter  becomes  fixed  by  the  cells  of  the 
central  nervous  system.  Failure  of  tetanus  antitoxin 
to  have  therapeutic  effects  is  due  in  most  cases  to 
faulty  administration.  Ashhurst  and  John  in  a  recent 
article  {Am,  Jour,  Med,  Sc,  June  and  July,  1913) 
present  what  is  undoubtedly  the  most  able  and  com- 
prehensive view  of  the  whole  subject  of  the  treatment 
of  tetanus.  Their  recommendations  will  be  followed 
here  to  a  great  extent. 

Antitetanic  serum  is  used  both  as  a  preventive  and 
a  curative  measure.  For  prophylactic  use,  in  the  case 
of  all  wounds  in  which  the  development  of  tetanus  is 
feared,  at  least  1500  units  of  the  antitoxin  should  be 
injected  as  early  as  possible  into  the  muscles  or  if 
possible  into  the  nerves  in  the  immediate  neighborhood 
of  the  wound.    The  longer  the  interval  that  is  allowed 


48  APPLIED  IMMUNOLOGY 

to  elapse  between  receipt  of  the  wound  and  injection 
of  the  serum,  the  larger  the  dose  of  antitoxin  required 
to  be  efficient.  Owing  to  the  fact  that  the  antitoxin  is 
all  eliminated  from  the  system  in  eight  or  ten  days 
after  the  injection,  and  that  dormant  tetanus  spores 
in  the  wound  may  develop  after  that  time,  it  is  ad- 
visable to  repeat  the  injection  during  the  second  week, 
and  if  possible  again  during  the  third  week.  In  the 
prophylaxis  of  tetanus,  however,  removal  of  any 
tetanus  bacilH  that  may  be  present  by  thorough  open- 
ing and  disinfection  of  the  woimd  is  of  greater  im- 
portance than  administration  of  antitoxin. 

Antitetanic  Serum  as  a  Curative  Measure, — The 
efficiency  of  tetanus  antitoxin  as  a  curative  agent  de- 
pends on  three  factors;  (1)  the  site  of  the  injection, 
(2)  thefrequency  of  the  injection,  (3)  the  quantity  of 
antitoxin  injected. 

(1)  The  Site  of  the  Injection, — Of  all  methods, 
the  subcutaneous  inoculation  of  tetanus  antitoxin  is 
least  efficient,  because,  as  Ashhurst  and  John  point 
out,  "  by  this  method  only  a  homoeopathic  dose  ulti- 
mately reaches  the  motor  nerves  through  which  the 
toxin  is  being  carried  to  the  spinal  cord,  while  by  far 
the  greater  part  of  the  antitoxin  is  distributed  to  the 
viscera,  where  it  can  be  of  no  possible  use.  Adminis- 
tered in  this  way,  overwhelming  amounts  are  required 
to  produce  any  effect."     Clinical  and  experimental 


ANTITOXIC  SERA  49 

evidence  points  overwhelmingly  to  intraspinal  (sub- 
dural) and  intraneural  injections  as  the  best  methods 
of  administering  tetanus  antitoxin,  for  by  these  means 
the  tissues  affected  by  the  toxin  are  reached  directly. 
Intravenous  injection  ranks  next,  as  by  this  means  all 
the  spinal  nerves  are  reached  at  once,  though  the  anti- 
toxin reaches  them  much  diluted. 

(2)  Frequency  of  Injection, — The  frequent  fail- 
ure of  tetanus  antitoxin  to  effect  a  cure  is  due  in  part 
to  the  fact  that  it  is  not  given  often  enough.  The  anti- 
toxin can  be  given  subcutaneously  every  three  hours, 
intravenously  once  or  twice  in  the  twenty-four  hours, 
intraspinally  and  intraneurally  every  day  if  necessary. 

(3)  Quantity  of  Antitoxin  Injected, — The  sheet- 
anchor  in  treatment  is  to  get  the  maximum  quantity 
of  antitoxin  indicated  into  the  patient's  body  as  soon 
as  possible.  One  of  the  authors  (B.  A.  T.),  in  the 
successful  treatment  of  a  case  of  tetanus,  reported  in 
Monthly  Cyclopcedia  and  Medical  Bulletin,  June, 
1911,  administered  213,740  units  of  antitetanic  serum. 
Of  this  enormous  and  unprecedented  quantity,  15,340 
units  were  injected  intraspinally,  the  remainder 
subcutaneously.  The  largest  inoculation  at  any  one 
time  was  35,400  units  and  the  maximum  quantity  in 
twenty-four  hours  was  97,940  units.  Subsequently, 
Ashhurst  and  John  state  that  subcutaneously,  in 
the  usual  acute  type  of  case,  at  least  100,000  units 


50  APPLIED  IMMUNOLOGY 

are  required  in  the  first  twenty-four  hours;  intra- 
venously, probably  15,000  to  25,000  units  should  be 
administered  at  first;  intraspinally,  from  3000  to  10,- 
000  units  should  be  given,  according  to  the  severity  of 
the  case.  The  writers  mentioned  recommend  for  in- 
traneural injections  as  great  amounts  as  the  nerves 
will  absorb^  ranging  from  750  to  1500  units.  For 
intraspinal  injection  the  same  technic  is  employed 
as  for  lumbar  puncture  and  spinal  angesthesia.  For 
intraneural  administration  the  motor  nerve  trunk 
supplying  the  region  of  the  wound  is  exposed  as  near 
to  the  spinal  cord  as  practicable,  and  the  antitoxin 
slowly  injected  directly  into  the  sheath. 

We  do  not  regard  it  as  out  of  place  here  to  quote 
in  full  Ashhurst  and  John's  recommendations  for  the 
rational  treatment  of  a  case  of  tetanus: 

The  patient  will  be  placed  in  quiet,  with  competent  nursing  facilities. 
As  soon  as  possible  after  coming  under  observation,  whether  this  be  in 
the  small  hours  of  the  night  or  at  bright  noon-tide,  the  motor  nerves  lead- 
ing from  the  wounded  part  will  be  exposed,  as  near  to  the  cord  as  practica- 
ble, and  as  much  antitoxin  as  each  will  contain  will  be  injected  toward 
the  spinal  cord.  An  intraspinal  injection  of  at  least  3000  units  will  then 
be  made  according  to  the  usual  technic  for  spinal  anaesthesia.  If  it  is 
possible  to  prick  the  cord  with  the  needle,  so  much  the  better.  Next  the 
wound  of  entrance  of  the  infection  will  be  widely  opened,  all  foreign 
bodies,  sloughs,  etc.,  will  be  removed  by  forceps,  scissors,  or  scalpel;  the 
wound  will  be  irrigated  with  hot  peroxide  of  hydrogen,  swabbed  out  with 
3  per  cent,  alcoholic  solution  of  iodine,  and  loosely  filled  with  gauze 
soaked  in  the  same  solution,  and  injection  of  antitoxin  will  be  made  (1500 
to  3000  units)  deeply  into  the  muscular  tissues  around  the  wound.  Con- 
tinuous proctoclysis,  as  used  in  cases  of  peritonitis,  will  be  given;  and  by 
mouth  or  in  the  rectal  fluid  will  be  administered  effective  doses  of  chloral 


ANTITOXIC   SERA  51 

and  bromides,  at  appropriate  intervals.^  Feeding  will  be  enforced,  by 
the  nasal  tube  passed  under  chloroform  anaesthesia,  if  necessary.  During 
the  course  of  the  first  day  a  moderate  amount  of  antitoxin  will  be  admin- 
istered intravenously;  probably  10,000  units  will  suffice. 

The  intraneural  and  intraspinal  injections  of  antitoxin  will  be  re- 
peated daily,  under  chloroform  anaesthesia,  until  marked  decrease  in 
spasticity  occurs.  Every  twelve  hours,  or  less  often,  a  moderate  amount 
of  antitoxin  will  be  injected  intravenously,  or  even  subcutaneously,  so  as 
to  neutralize  the  circulating  toxins;  but  the  main  reliance  will  be  placed 
on  intraneural  and  intraspinal  injections.  The  administration  of  spinal 
depressants  will  be  continued  as  long  as  they  are  indicated;  a  comatose 
state  or  muscular  relaxation  naturally  are  contra-indications.  The  wound 
will  be  dressed  daily,  as  above  described,  until  a  healthy  granulating  sur- 
face is  obtained. 

The  administration  of  cardiac,  pulmonary  and 
renal  stimulants  to  meet  the  particular  conditions,  is 
invariably  a  matter  of  necessity. 

The  application  of  the  treatment  as  above  outlined 
and  commenced  within  twelve  hours  of  the  onset  of 
symptoms,  should  reduce  the  mortality  of  tetanus  to 
less  than  20  per  cent. 

Antigonococcic  Serum. — Attempts  have  been 
made  by  Torrey  and  others  to  produce  an  antitoxic 
serum  by  injecting  a  toxin  separated  by  filtration  from 
cultures  of  gonococci.  But  it  has  been  found  that 
while  this  substance  is  toxic  for  laboratory  animals,  it 
does  not  produce  antibodies  that  render  the  animal  in- 

^  The  authors  feel  from  their  experience  that  complete  reliance  should 
not  be  placed  on  these  drugs,  and  recommend  intraspinal  injections  of 
chemically  pure  magnesium  sulphate  in  25  per  cent,  solution  to  allay  mus- 
cular spasm  and  convulsive  seizures,  in  quantities  of  2  to  5  c.c,  the  maxi- 
mum being  1  c.c.  for  each  25  pounds  of  body  weight.  Should  signs  of 
respiratory  failure  supervene,  chloretone  administered  by  mouth  or  by 
rectum,  preferably  the  latter  in  one  drachm  doses,  may  be  substituted  for 
or  alternated  with  the  injections  of  magnesium  sulphate. 


52  APPLIED  IMMUNOLOGY 

jected  immune  to  the  toxin.  Efforts  to  produce 
immune  sera  by  injections  of  gonococci  themselves 
have  been  more  successful,  and  will  be  discussed  under 
antibacterial  sera  (Chapter  VII). 

Antituberculosis  Serum. — IMariagliano  has  pro- 
duced a  serum  by  immunizing  horses  with  toxins  made 
from  the  filtrate  of  cultm-es  of  tubercle  bacilli.  His 
favorable  results  have  not  been  generally  confirmed, 
and  antiserum  is  little  used  in  tuberculosis. 

Antidysenteric  Serum, — The  dysentery  bacilli  of 
the  Shiga  type  form  an  extracellular  toxin  and  from 
this  it  is  possible  to  prepare  a  true  antitoxic  serum. 
The  toxin,  together  with  antidysenteric  serum,  is  in- 
jected into  the  horse  in  gradually  increasing  doses. 
Killed  cultures  of  the  bacilli  are  also  injected  to  give 
the  serum  antibacterial  properties.  The  standard 
strength  of  the  antiserum  is  such  that  0.5  c.c.  will  pro- 
tect a  1500-gramme  rabbit  against  three  times  the 
smallest  fatal  dose  of  toxin  (Schorer).  In  Japan  by 
the  use  of  this  serum,  the  mortality  of  dysentery  of  the 
Shiga  type  has  been  reduced  from  28-37  per  cent,  to 
8-12  per  cent.  In  mild  cases  one  dose  of  10  c.c.  of 
serum  is  injected,  in  severer  cases  two  injections  of 
10  c.c.  are  made  six  to  ten  hours  apart,  never  more 
than  20  c.c.  in  one  day.  Dysentery  caused  by  bacilli 
of  the  Shiga  type  is  rare  in  the  United  States,  being 
largely  confined  to  tropical  and  semitropical  coun- 


ANTITOXIC   SERA  53 

tries.  The  form  of  bacillary  dysentery  met  with  in 
the  United  States  is  usually  due  to  the  mannite- fer- 
menting or  Flexner  type  of  organism,  which  will  be 
dealt  with  in  the  chapter  on  antibacterial  sera. 

Antibotulism  Serum. — Meat  poisoning  is  due  in 
many  cases  to  a  toxin  produced  by  the  Bacillus  botu- 
linus.  This  organism  is  anaerobic,  and  depends  for  its 
action  upon  an  extracellular  toxin.  This  toxin  can  be 
obtained  artificially  from  bouillon  cultures  of  the  or- 
ganism. The  Institute  for  Infectious  Diseases  in 
Berlin  has  produced  an  antitoxin  by  immunization  of 
animals,  but  data  are  still  insufficient  to  pass  judg- 
ment upon  its  curative  properties. 

Antiphytotoocic  Serum, — Many  forms  of  hay  fever 
are  manifestations  of  anaphylaxis  or  hypersensibility 
to  the  pollen  of  certain  plants,  such  as  golden-rod,  rag- 
weed, honeysuckle,  chrysanthemum,  etc.  Dunbar  has 
produced  an  antitoxin,  known  as  "  pollantin,"  by  in- 
jecting horses  with  extracts  from  various  pollens.  The 
antitoxin  is  put  up  in  liquid  form  for  inoculations,  in 
powder  form  for  local  application.  In  certain  cases  the 
antitoxin  gives  protection  and  relief,  but  as  a  rule  only 
for  a  limited  time.  Its  use  is  occasionally  attended 
by  severe  symptoms  of  anaphylaxis,  so  that  caution 
must  be  observed  in  beginning  treatment  with  it. 

Antivenin, — Antivenin  is  an  antitoxic  serum  pre- 
pared to  counteract  the  effects  of  snake  poison.    The 


54  APPLIED  IMMUNOLOGY 

nature  and  mode  of  action  of  snake  venoms  have  been 
studied  extensively,  especially  by  Calmette  and  Flex- 
ner  and  Noguchi.  Their  researches  have  shown  that 
poisonous  snakes  can  be  classified  into  two  broad 
groups,  according  to  the  effects  produced  by  the 
toxins  of  their  venom.  To  one  group  belong  the 
cobras,  whose  venom  is  principally  in  the  nature  of  a 
neurotoxin,  acting  especially  on  the  respiratory  centre 
in  the  medulla,  while  in  the  other  group,  containing 
the  vipers  and  rattlesnakes,  the  venom  contains  a 
hemorrhagin,  and  causes  extravasation  of  blood  in 
various  regions  of  the  body.  In  addition,  all  snake 
venoms  possess  hsemolytic  properties,  found  most 
markedly  in  cobra  venom.  Against  the  neurotoxin 
of  cobra  venom,  Calmette  has  successfully  produced 
an  antitoxin  (antivenin)  by  injecting  horses  with  the 
venom.  This  is  only  of  therapeutic  value,  however, 
against  cobra  bites,  and  is  useless  as  an  antidote 
for  rattlesnake  bites.  Attempts  have  been  made,  but 
unsuccessfully  so  far,  to  produce  a  reliable  antihemor- 
rhagic  serum  for  the  treatment  of  rattlesnake  bites. 

The  antivenin  of  Calmette  may  be  injected  in 
doses  of  10  to  20  e.c.  To  be  of  any  value  it  naturally 
must  be  used  as  soon  as  possible  after  the  bite  is  re- 
ceived, and  is  probably  useless  after  3  or  4  hours. 
The  serum  has,  therefore,  only  limited  application  as 
a  therapeutic  measure. 


VII 

ANTIBACTERIAL  SERA 

It  has  been  experimentally  possible  to  produce 
antibacterial  sera  for  a  much  larger  number  of  organ- 
isms than  antitoxic  sera,  though  in  the  case  of  none  of 
them  have  such  brilliant  therapeutic  results  been  ob- 
tained as  in  the  case  of  diphtheria  and  tetanus  anti- 
toxin. Yet  in  infections  by  a  few  organisms,  notably 
the  staphylococcus,  streptococcus,  pneumococcus,  gon- 
ococcus,  meningococcus,  typhoid  bacillus,  colon  bacil- 
lus, dysentery  bacillus,  cholera  vibrio,  plague  bacillus 
and  anthrax  bacillus,  antibacterial  sera  of  consider- 
able therapeutic  value  have  been  produced. 

Antistaj^hylococcic  Serum, — Various  attempts 
have  been  made  on  horses  and  other  animals  to  pro- 
duce a  potent  antistaphylococcic  sermn,  but  they  have 
almost  invariably  resulted  unsuccessfully,  although 
Doyen  and  Paltchikowsky  assert  that  they  have  suc- 
ceeded partially.  Schorer  states  that  the  value  of  the 
serum  is  inconsiderable,  and  its  injection  in  the  treat- 
ment of  staphylococcus  infections  is  seldom  or  never 
warranted.  Such  noted  authorities  as  Ehrlich,  Bordet 
and  Citron  omit  even  to  mention,  in  their  works  on 
immunity,  the  existence  of  antistaphylococcic  serum. 
The  failure,  or  only   partial   success,   in  producing 

55 


56  APPLIED  IMMUNOLOGY 

potent  antistaphylococcic  serum  has  possibly  been  due 
to  utilization  of  avirulent  or  univalent  cultures  of  the 
organisms.  One  of  the  most  important  factors  con- 
ducive to  the  successful  production  of  this  serum  is  the 
employment  of  staphylococcus  cultures  from  many 
sources.  With  this  essential  in  mind,  one  of  us 
(Thomas)  isolated  eighteen  strains  of  Micrococcus 
aureus  in  pure  culture  from  many  sources,  as  fur- 
uncles, carbuncles,  abscesses  of  the  scalp,  thoracic 
empyema,  axillary  abscess,  and  septicaemia.  From 
these  strains  24-hour  cultures  were  grown  and  from 
the  mixed  growth  a  suspension  in  physiologic  salt 
solution  was  prepared  containing  32,400,000,000  cocci 
to  the  cubic  centimetre.  This  was  then  heated  for  one 
hour  over  a  water-bath  at  60°  C.  The  animal  selected 
for  the  purpose  of  immunization  was  a  full-grown  ram 
weighing  165  pounds.  The  degree  of  immunity  of 
the  animal  was  governed  by  determination  of  the 
opsonic  index  for  the  polyvalent  staphylococcic  sus- 
pension (see  Fig.  3).  At  the  first  injection  the  ram 
received  intraperitoneally  972,000,000,000  dead  sta- 
phylococci. Thereafter  at  weekly  intervals  either  grad- 
ually increasing  doses,  or  slightly  smaller  doses  heated 
for  a  shorter  time,  and  finally  unheated  organisms,  were 
injected.  After  twelve  inoculations,  the  index  was 
found  to  be  2.6,  and  the  animal  was  bled  to  death 
from  the  carotid  artery.    The  serum  was  hermetically 


ANTIBACTERIAL  SERA  57 

sealed  in  glass  ampoules  containing  1  and  2  c.c.  each. 
The  appended  chart  (Fig.  3)  shows  the  inoculations 
and  curve  of  opsonic  indices  illustrating  the  immunity 
of  the  ram. 

From  therapeutic  employment  of  this  antistaphy- 
lococcic  serum  in  many  cases  of  carbuncles  and  fur- 
uncles, in  doses  ranging  from  1  to  6  c.c,  the  following 
conclusions  were  drawn. 

1.  The  antistaphylococcic  serum  as  herein  pre- 
pared and  described  possessed  unquestionable  thera- 
peutic efficiency  in  a  series  of  conditions,  both  general 
and  local,  due  to  infections  by  the  Micrococcus  aureus, 

2.  Biologic  therapy  by  a  potent  polyvalent  anti- 
staphylococcic serum  is  more  effective  in  the  presence 
of  a  staphylococcic  bacterisemia  than  is  the  correspond- 
ing autogenous  bacterin. 

3.  By  virtue  of  the  more  immediate  and  decisive 
effects  of  the  antiserum,  it  deserves  first  choice  over 
the  bacterin  in  the  treatment  of  furunculosis  and  car- 
bunculosis;  on  the  other  hand,  a  more  intensive  and 
lasting  immunity  can  be  conferred  on  the  individual 
by  supplementing  the  serum  with  two  or  three  inocula- 
tions of  the  autogenous  bacterin. 

4.  It  is  to  be  regretted  that  no  attempt  was  made 
to  standardize  this  antiserum  with  respect  to  standard 
units,  since  it  must  be  conceded  that  the  therapeutic 
failure  or  inefficiency  of  many  serums  is  referable  to 


58  APPLIED  IMMUNOLOGY 

the  deficiency  of  the  immune-body  content  of  that 
particular  serum,  or  in  other  words,  to  an  improper  or 
incomplete  immunization  of  the  animal  utilized  for  the 
production  of  the  antiserum. 

Antistreptococcic  Serum, — Various  antistrepto- 
coccic sera  have  been  employed  for  some  time  with 
more  or  less  success.  They  are  nearly  all  made  by  in- 
jecting horses  with  increasing  doses  of  killed  strepto- 
cocci recovered  from  different  lesions,  thus  securing 
polyvalent  antisera.  Standardization  of  the  strength 
of  these  sera  is  difficult,  the  initial  dose  being  more  or 
less  empirical,  and  succeeding  doses  being  guided  by 
clinical  effects  observed. 

The  serum  is  indicated  in  all  streptococcic  infec- 
tions, particularly  in  septicaemia,  where  rapid  effects 
are  desirable,  and  where  bacterial  vaccines  are  not  suit- 
able. The  dose  varies  from  10  to  100  c.c,  to  be 
repeated  in  accordance  with  the  symptomatic 
indications. 

Antipneumococcic  Serum, — Passive  immunization 
in  pneumococcus  infections  has  been  attempted  by 
means  of  antibacterial  serum  obtained  from  horses 
previously  injected  with  different  strains  of  pneumo- 
cocci.  This  serum  depends  for  its  action  chiefly  upon 
an  increased  production  of  bactericidal  and  opsonizing 
substances.  In  pneumonia  the  results  of  its  use  by 
most  observers  have  been  disappointing,  the  dose  of 


ANTIBACTERIAL  SERA  59 

serum  ranging  from  10  to  20  c.c.  in  this  disease.  Cole 
finds  the  results  of  treatment  of  pneumonia  by  anti- 
pneumococcic  serum  to  be  encouraging  in  certain 
cases,  when  used  in  large  doses  (100  c.c.  or  more). 
He  states  that  this  action  is  probably  in  part  due  to 
antitoxic  substances.  In  other  pneumococcus  infec- 
tions, especially  serpiginous  ulcer  of  the  cornea,  bene- 
fit from  its  use  has  been  seen. 

Antigonococcic  Serum. — Torrey  and  Rogers  have 
produced  an  antibacterial  serum  that  is  of  considerable 
therapeutic  value  in  certain  gonococcus  infections. 
This  serum  is  made  by  injecting  strong,  full-gro^vn 
rams  with  24-hour  polyvalent  cultures  of  virulent 
gonococci.  Nine  or  ten  in j  ections  are  usually  required 
to  produce  a  serum  of  high  potency,  the  bacterial  sus- 
pension being  heated  for  half  an  hour  at  65°  C.  be- 
fore the  first  two  or  three  injections,  after  which  the 
unheated  gonococci  are  used.  When  sufficiently  im- 
munized the  animal  is  bled  from  the  carotid  artery, 
the  serum  separated,  and  stored  in  sterile  ampoules. 
Standardization  of  this  serum  in  units  is  difficult,  but 
some  idea  of  its  strength  may  be  gained  by  testing  its 
agglutinating  power.  The  dose  of  the  antigonococcic 
serum  varies  from  2  to  6  c.c,  injected  under  the  skin, 
and  repeated  every  few  days.  The  serum  is  only  to  be 
used  as  an  adjunct  to  other  methods  of  treatment.  It 
is  of  little  or  no  value  in  acute  and  chronic  urethritis. 


eo  APPLIED  IMMUNOLOGY 

prostatitis,  and  conjunctivitis,  but  has  beneficial  ef- 
fects in  cases  of  gonorrhoeal  arthritis,  endocarditis, 
peritonitis,  and  septicaemia. 

Antimeningococcic  Serum, — Kolle  and  Wasser- 
mann,  Jochman,  and  others  have  succeeded  in  immu- 
nizing animals  by  injections  of  the  Diplococcus  intra- 
cellularis  of  Weichselbaum,  the  antisera  thus  obtained 
proving  of  considerable  value  in  meningitis  due  to  this 
organism.  The  serum  of  Flexner  and  Jobling,  how- 
ever, both  on  account  of  its  potency  and  also  the 
method  of  its  administration,  has  superseded  all  others. 
Flexner  and  Jobling  prepare  their  serum  by  inject- 
ing into  horses,  first,  gradually  increasing  doses  of 
heated  meningococci,  followed  by  injections  of  un- 
heated  organisms,  and  finally  injections  of  an  autoly- 
sate or  extract  of  the  meningococci  which  contains  the 
endotoxin.  The  serum  thus  produced  acts  both  by 
opsonizing  and  bacteriolytic  properties.  Accurate 
standardization  of  the  serum  is  not  possible,  the  dose 
depending  largely  upon  clinical  indications.  Flexner 
and  Jobling  administer  the  serum  by  subdural  injec- 
tion, a  lumbar  puncture  being  first  done  and  some  of 
the  spinal  fluid  removed.  The  quantity  of  serum  to  be 
injected  ranges  from  30  to  60  c.c.  In  severe  cases  the 
injection  must  be  repeated  in  12  or  24  hours  and  there- 
after as  long  as  meningococci  are  found  in  the  spinal 
fluid.    By  means  of  this  senmi,  the  mortality  of  epi- 


ANTIBACTERIAL  SERA  61 

demic  cerebrospinal  meningitis  due  to  the  Diplococcus 
intracellularis  has  been  reduced  from  70  per  cent,  to 
about  30  per  cent. 

The  antimeningococcic  serum  may  also  be  used  as 
a  preventive  measure  in  persons  exposed  to  the  disease. 

Antityphoid  Serum. — Antibacterial  sera  have  been 
prepared  by  immunizing  horses  by  injections  of  mixed 
strains  of  killed  and  live  typhoid  bacilli.  The  serum 
thus  produced  has  been  injected  into  persons  suffering 
from  typhoid  fever,  in  daily  doses  of  10  c.c.  or  more. 
So  far  the  results  have  been  disappointing  either  in 
shortening  the  disease  or  lessening  the  severity  of  the 
symptoms.  For  protective  purposes,  active  immu- 
nization by  injection  of  killed  cultures  of  typhoid 
bacilli  has  proved  more  successful. 

Anticolonic  Serum. — Hans  ]Much  finds  that  nor- 
mal blood-serum  has  a  bactericidal  action  upon  certain 
strains  of  colon  bacilli,  while  on  other  strains  blood- 
plasma  has  this  action  but  not  blood-serum.  He  has 
employed  normal  blood-plasma  and  serum  locally  in 
cystitis  caused  by  infection  with  the  colon  bacillus  with 
some  success.  Much  has  also  been  able  to  produce  a 
powerful  specific  antiserum  by  injecting  animals  with 
several  strains  of  colon  bacilli,  which  have  bacteriolytic 
properties  against  all  strains  of  colon  bacilli.  He  has 
used  this  also  with  benefit  as  a  local  application  in 
colon  bacillus  pyelitis  and  cystitis. 


62  APPLIED  IMMUNOLOGY 

Antidy  sent  eric  Serum, — We  saw  that  an  antitoxic 
serum  has  been  successfully  produced  and  used  in  in- 
fections with  the  Shiga  type  of  dysentery  bacillus. 
The  form  of  dysentery  more  common  in  the  United 
States  is  due  to  the  mannite-fermenting  or  Flexner 
type  of  organism.  An  antibacterial  serum  has  been 
obtained  for  treatment  of  infection  with  this  organism, 
but  used  with  indifferent  success. 

Anticholera  Serum, — Attempts  have  been  made  to 
obtain  both  antitoxic  and  antibacterial  sera  for  use  in 
cholera.  Kolle  and  Wassermann,  MetchnikofF  and 
Roux  tried  to  get  a  soluble  toxin  from  cultures  of  the 
vibrio,  and  from  that  to  make  an  antitoxin.  But  it  is 
probable  that  they  only  obtained  the  intracellular 
toxin  from  disintegration  of  the  organisms.  The  sera 
produced  by  inoculation  of  animals  with  these  endo- 
toxins or  with  the  bacteria  themselves,  have  been  of 
little  value  in  the  treatment  of  cholera.  More  favor- 
able results  have  followed  active  immunization  with 
cultures  of  the  organisms. 

Antiplague  Serum. — Two  types  of  antiplague 
serum  have  been  employed.  The  first  is  made  by  in- 
oculation of  horses  with  plague  bacilli,  and  the  other 
by  inoculation  with  nucleo-proteids  produced  from 
cultures.  Extensive  trials  of  these  sera  in  India  show 
little  benefit  from  their  use  in  the  treatment  of  plague, 
though  life  may  be  slightly  prolonged  by  them. 


ANTIBACTERIAL  SERA  63 

Anti-anthrcur  Serum, — Of  the  many  antisera  that 
have  been  produced  for  the  treatment  of  anthrax,  that 
of  Sclavo  is  the  best.  This  is  prepared  by  inoculation 
of  asses  simultaneously  with  cultures  of  the  Bacillus 
anthracis  and  antiserum,  a  very  powerful  serum  being 
thus  obtained.  Sclavo  advises  a  dose  of  30  to  40  c.c. 
divided  into  three  or  four  parts,  injected  into  the  ab- 
dominal wall  in  different  regions.  This  dose  may  be 
repeated  in  24  hours  if  necessary.  In  grave  cases,  in- 
travenous injection  of  the  serum  is  recommended. 
Statistics  tend  to  show  that  use  of  the  serum  reduces 
the  mortality  of  anthrax  from  about  26  per  cent,  to 
6  per  cent. 

Antimelitensis  Serum, — A  curative  serum  for  the 
treatment  of  Malta  or  Mediterranean  fever  was  first 
prepared  by  Wright  by  immunizing  goats  with  cul- 
tures of  M,  meliteims.  Horses  were  later  employed 
for  immunization  with  more  or  less  encouraging  re- 
sults. However,  the  method  of  passive  immunization 
in  this  disease  has  been  overshadowed  in  recent  years 
by  active  immunization. 


VIII 

MISCELLANEOUS  SERA  AND  EXTRACTS 

ANTIRABIC  SERUM— ANTILEPROSY  SERUM— ANTITYPHOID 
EXTRACT  OF  JEZ— LEUCOCYTIC  EXTRACT— ANTIFER- 
MENT  —  ANTICARCINOM  ATOUS  EXTRACTS  —  PYOCYA- 
NASE— ANTITHYROID  SERUM  AND  EXTRACTS— SPANG- 
LER'S  CROTALIN— PHYLACOGENS 

There  are  several  extracts  and  sera  that  have  been 
introduced  as  methods  of  treatment  in  various  diseases, 
but  which  require  httle  more  than  brief  mention,  either 
because  they  have  not  yet  been  given  thorough  trial, 
or  because  results  with  them  have  not  been  sufficiently 
striking  as  to  establish  them  as  routine  measures. 

Antirabic  Serum. — The  success  of  active  immu- 
nization in  the  prevention  of  hydrophobia  has  led 
several  workers  to  attempt  to  obtain  a  sermn  for  pas- 
sive immunization  in  this  disease.  Such  immunity  has 
been  experimentally  produced  by  means  of  serum 
from  dogs  that  have  been  actively  immunized  by  in- 
jections of  rabies  virus.  The  practical  value  of  the 
serum,  however,  is  not  yet  sufficiently  established. 
The  recent  announcement  of  the  isolation  and  arti- 
ficial culture  of  the  rabies  organism  by  Noguchi  leads 
to  the  hope  of  success  along  this  line. 

Antileprosy  Serum. — As  yet,  it  has  not  been  pos- 
sible to  cultivate  the  leprosy  bacillus  on  artificial 
64 


MISCELLANEOUS  SERA  AND  EXTRACTS        65 

media,  a  fact  that  has  retarded  considerably  any  re- 
search on  immunization  in  leprosy.  Passive  immu- 
nization has  been  attempted  by  Carrasquilla,  Herman 
and  Abraham,  and  others,  by  injecting  the  blood  of 
lepers  or  the  juice  from  nodular  lesions  into  the  horse, 
and  employing  the  serum  from  the  animal  thus  in- 
jected in  the  treatment  of  the  disease.  Slight  im- 
provement has  been  reported  in  a  few  cases. 

Antityphoid  Ecctract  of  Jez. — Jez  claimed  anti- 
toxic properties  from  an  extract  prepared  from  the 
bone-marrow,  spleen,  and  lymph-nodes  of  an  animal 
immunized  to  typhoid  bacilH.  This  has  been  found  to 
have  but  little  value  in  the  treatment  of  typhoid  fever. 

Leiicocytic  Extract, — Attempts  have  been  made 
to  increase  the  phagocytic  powers  by  preparing  and 
injecting  extracts  made  from  leucocji;es,  especially 
in  pneumonia.  Both  human  and  lower  animal  ex- 
tracts have  been  employed.  Manoukhine  uses  leuco- 
cytes from  the  patient's  owti  blood,  removing  them 
from  7  c.c.  of  the  blood  by  centrifuging.  He  kills  the 
leucocytes  by  freezing,  and  then  suspends  them  in  1 
c.c.  of  salt  solution,  which  he  injects.  He  claims  to 
have  obtained  beneficial  results  in  pneumonia  by  this 
method. 

Antiferment. — The  antiferment  treatment  of  in- 
fections was  introduced  by  A.  Miiller  in  1907.  In  the 
destruction  of  the  polymorphonuclear  leucocj^tes  dur- 


66  APPLIED  IMMUNOLOGY 

ing  suppuration  there  is  liberated  from  them  a  pro- 
teolytic ferment  known  as  proteolysin  which  has  the 
power  of  dissolving  the  albumin  of  the  tissues  and 
thus  breaking  down  the  limiting  wall  of  the  abscess 
and  allowing  it  to  extend.  Theoretically,  therefore, 
much  good  would  be  accomplished  if  this  tissue  de- 
struction could  be  inhibited.  Normal  blood-sermn  con- 
tains antiferments  which  neutralize  the  proteolysis  of 
the  leucocytes,  but  this  under  most  conditions  of  in- 
flammation is  unable  in  sufficient  quantity  to  reach 
the  tissue  that  is  being  broken  down.  JVItiller  there- 
fore reconmiended  the  direct  introduction  of  the  anti- 
ferment  in  large  amount  by  injecting  normal  serum 
into  the  inflamed  area,  or  by  packing  abscess  cavities, 
after  evacuation  of  the  pus,  with  gauze  saturated  with 
the  serum.  The  patient's  own  blood-serum  or  ascitic 
or  hydrocele  fluid  is  recommended  for  the  purpose. 
Where  it  is  impractical  to  obtain  these,  normal  horse 
serum  can  be  substituted.  While  this  form  of  treat- 
ment has  some  theoretical  justification,  practically  it 
has  not  met  with  any  notable  success.  The  injection 
of  acute  abscesses  is  not  unattended  with  danger  of 
anaphylaxis.  Irrigation  of  abscess  cavities  or  pack- 
ing with  gauze  saturated  with  the  fluid  may  in  some 
cases  be  a  useful  adjunct  to  the  ordinary  surgical 
measures. 


MISCELLANEOUS   SERA  AND  EXTRACTS        67 

Anticarcinomatous  Extracts. — Extracts  have  been 
prepared  from  various  normal  and  abnormal  animal 
tissues  for  use  in  the  treatment  of  cancer.  While  cer- 
tain of  these  gave  great  promise  at  first  and  ap- 
parently brought  about  cures,  none  of  them  so  far  have 
stood  the  test  of  time.  Again,  certain  substances  that 
have  given  successful  results  in  the  cure  of  the  arti- 
ficially implanted  malignant  tumors  in  lower  animals, 
have  not  had  the  same  effects  in  corresponding  disease 
in  man.  Among  non-specific  products  that  have  been 
tried  for  this  purpose  may  be  mentioned  extracts  of 
thymus  and  thyroid  glands,  spleen,  and  pancreas. 
Coca  and  Gilman  prepared  emulsions  from  human 
carcinomata  and  injected  them  into  patients  suffering 
from  growths  of  similar  histology.  This  method  was 
hailed  at  first  as  the  long-looked- for  specific  non-surgi- 
cal treatment  for  cancer,  but  has  not  lived  up  to  ex- 
pectations. The  injection  was  supposed  to  stimulate 
the  formation  in  the  patient's  blood  of  substances  capa- 
ble of  dissolving  the  tumor  tissue.  Recognizing  that 
malignant  tumors  are  analogous  in  histological  and 
physiological  characteristics  to  embryonic  tissues, 
Fichera  {Lancet,  Oct.,  1911)  announced  the  results  of 
his  experiments  with  autolysates  of  fetal  tissues  in 
patients  suffering  from  carcinoma.  Of  36  patients,  in 
18  the  results  were  inconclusive,  in  8  there  was  no 


68  APPLIED  IMMUNOLOGY 

marked  benefit,  in  5  there  was  distinct  benefit,  while 
5  were  apparently  cured.  The  autolysates  were  made 
by  mincing  fetal  and  embryonic  tissues,  placing  them 
in  normal  salt  solution  and  incubating  for  two  months. 
The  clear  fluid  was  then  injected  subcutaneously  or 
directly  into  the  neoplasm.  Recently,  Babcock  {In- 
ternational Clinics^  Vol.  II,  23rd  Series,  1913)  has 
reported  a  trial  of  fetal  autolysates  prepared  by 
Fichera's  method  in  21  cases  of  recurrent  or  inoper- 
able cancer,  and  concludes  that  this  method  is  of  no 
distinct  value  in  the  treatment  of  malignant  disease  in 
man.  It  would  seem,  therefore,  that  Fichera's  treat- 
ment is  doomed  to  the  same  failure  that  has  so  far 
attended  all  other  attempts  at  cure  of  carcinoma  by 
organo-  or  serum  therapy. 

Pyocyanase, — Pyocyanase  is  a  ferment  isolated 
from  cultures  of  B,  pyocyaneus,  said  to  have  high  di- 
gestive powers  for  proteids.  It  has  been  employed 
in  the  treatment  of  diphtheria,  the  protein  digestive 
action  facilitating  the  removal  of  the  membrane.  Pyo- 
cyanase has  also  been  used  in  gonorrhoeal  urethritis, 
in  follicular  tonsillitis  and  as  a  spray  for  disinfection 
of  the  nose. 

Antithyroid  Serum  and  E  attracts, — Working 
upon  the  established  theory  that  the  symptoms  of  ex- 
ophthalmic goitre  were  due  to  a  hypersecretion  of  the 


MISCELLANEOUS  SERA  AND  EXTRACTS        69 

thyroid  gland,  investigators  have  attempted  to  formu- 
late a  method  of  treatment  upon  rational  lines  of  or- 
ganotherapy. In  1899  Otto  Long  employed  the  milk 
of  thyroidectomized  goats,  believing  that  this  pos- 
sessed some  property  antagonistic  to  the  gland  secre- 
tion. Under  the  name  of  rodagen,  a  powder  was  pre- 
pared from  the  milk  of  thyroidectomized  goats,  50  per 
cent,  of  which  was  composed  of  the  active  constituent 
of  the  milk  and  50  per  cent,  sugar  of  milk.  The  dose 
of  this  is  1  to  3  drachms  a  day.  Considerable  improve- 
ment was  noted  following  its  use  in  some  cases,  but  no 
disappearance  of  the  exophthalmos,  the  goitre,  or  the 
tachycardia.  Others  have  employed  antithyroid  serum 
from  the  blood  of  thyroidless  sheep  in  doses  of  1  to  5 
c.c.  daily.  This  had  no  conspicuous  effect.  Thyroi- 
dectin,  a  powder  consisting  of  the  desiccated  blood  of 
thyroidectomized  animals,  has  also  been  used,  it  is 
claimed,  with  marked  benefit  in  some  cases  of  exoph- 
thalmic goitre.  The  dose  is  one  or  two  5-grain  cap- 
sules three  times  a  day.  None  of  these  preparations 
can  be  said  to  have  fulfilled  expectations. 

Crotalin  in  Epilepsy, — The  apparent  improve- 
ment in  an  epileptic  after  being  bitten  by  a  rattlesnake 
led  Spangler  to  treat  a  number  of  epileptics  by  injec- 
tions of  increasing  doses  of  rattlesnake  poison  or  cro- 
talin.   Spangler  reported  a  certain  number  of  favora- 


70  APPLIED  IMMUNOLOGY 

ble  results,  which,  however,  have  not  been  confirmed 
by  others.  The  irrational  basis  for  this  treatment 
and  the  dangerous  nature  of  the  poison  are  such  as  to 
make  its  use  unjustifiable.  Among  others,  Yawger 
(Penna.  Med.  Jour,,  Sept.,  1914,  p.  964) ,  after  a  trial 
of  crotalin  in  six  cases  of  idiopathic  epilepsy,  gives  the 
results  of  his  experience  as  follows :  "  Two  patients 
were  uninfluenced;  two  were  worse  during  the  treat- 
ment; one,  early  in  the  course,  developed  such  in- 
tolerant toxic  symptoms  that  further  experimentation 
was  im justified,  and  the  last  patient  died  two  and  a 
half  months  after  treatment.  While  we  did  not  feel 
that  death  resulted  from  the  use  of  crotalin,  the  pa- 
tient's disease  certainly  was  not  benefited  by  the 
treatment." 

Phylacogens. — Within  the  last  year  or  two  con- 
siderable exploitation  has  been  given  to  certain  bac- 
terial products  for  the  treatment  of  infections  of  all 
kinds,  under  the  name  of  phylacogens.  It  is  claimed 
by  the  manufacturers  that  these  "  modified  bacterial 
derivatives  '*  are  superior  to  bacterial  vaccines  as  ordi- 
narily prepared.  Phylacogens  are  sterile  aqueous 
solutions  of  bacterial  derivatives  prepared  by  growing 
the  bacteria  in  artificial  culture  media,  killing  them  by 
heat,  and  then  removing  their  soluble  products  by 
filtration.    In  preparing  special  phylacogens  for  the 


MISCELLANEOUS  SERA  AND  EXTRACTS        71 

treatment  of  various  diseases,  the  mixed  products  of 
several  varieties  of  bacteria  are  employed,  whether 
these  varieties  of  bacteria  are  found  in  the  particular 
infection  or  not. 

Without  going  into  further  detail,  we  are  of  the 
opinion  that  the  therapeutic  use  of  phylacogens  is 
based  upon  erroneous  conceptions  of  infection  and  im- 
munity, is  unscientific,  empirical,  and  unjustified. 
Moreover,  there  is  evidence  that  their  administration 
is  not  unattended  with  risk  to  the  patient. 


IX 

AGGLUTININS 

THE  WIDAL  PHENOMENON  AND  OTHER  AGGLUTINATION 

REACTIONS 

Agglutinins  are  antibodies  belonging  to  the 
second  order  of  Ehrlich  (see  Chapter  III).  They 
are  formed  in  the  blood-serum  as  a  result  of  infection 
with  specific  bacteria.  Agglutinins  have  the  power 
of  causing  the  specific  bacteria,  in  response  to  which 
they  have  been  called  forth,  to  lose  their  motility  and 
to  be  drawn  together  in  clumps.  Agglutinins  are 
probably  not  very  important  factors  in  immunity,  but 
are  of  great  value  for  diagnostic  pm'poses,  as  their  ac- 
tion can  be  studied  in  vitro.  Thus  blood-serum  in 
various  dilutions,  when  brought  in  contact  with  a  cul- 
ture of  the  particular  organism  that  has  infected  the 
patient,  will  after  a  time  cause  loss  of  motility  and 
clumping  of  the  bacteria  in  the  culture.  The  principal 
infections  in  which  agglutination  tests  have  proved 
most  serviceable  in  diagnosis  are  those  caused  by  ty- 
phoid, paratyphoid,  dysentery,  and  colon  bacilli. 
These  tests  are  of  great  value,  sometimes  in  differen- 
tiating closely  related  organisms.  Agglutinins  be- 
long to  what  are  known  as  "  group-reaction  "  anti- 
bodies, that  is  to  say,  they  act  upon  closely  allied 

72 


AGGLUTININS  7S 

members  of  the  same  group  of  bacteria,  such  as  B, 
typhosus,  and  B,  paratyphosus  A  and  B,  though  not 
with  the  same  intensity.  Members  of  this  group  can 
be  differentiated  by  using  different  dilutions  of  the 
agglutinating  serum,  the  specific  organism  involved 
being  agglutinated  by  the  greatest  dilution  of  serum. 
Agglutination  tests  are  occasionally  employed  in  the 
diagnosis  of  glanders,  cholera,  Malta  fever,  and  in 
staphylococcus  and  streptococcus  infections. 

The  Widal  Reaction 

The  agglutination  test  for  the  diagnosis  of  typhoid 
fever  is  known  generally  as  the  Widal  reaction,  be- 
cause this  investigator,  though  by  no  means  the  dis- 
coverer of  the  method,  first  used  it  extensively  in 
clinical  work. 

There  are  two  methods  of  carrying  out  the  test: 
microscopically  and  macroscopically.  Of  these,  the 
microscopic  method  is  that  more  generally  employed. 

A.       MICROSCOPIC   METHOD 

For  the  performance  of  the  test  we  require  blood- 
serum  from  the  suspected  patient,  and  a  recent  culture 
of  typhoid  bacilli. 

The  description  of  the  test  may  therefore  be 
divided  into  three  portions,  viz.:  {a)  collection  and 
dilution  of  the  patient's  blood-serum;   (b)   prepara- 


74  APPLIED  IMMUNOLOGY 

tion  of  the  culture  of  typhoid  bacilH ;  ( c )  teclmic  of  the 
test. 

(a)  Cpllection  and  Dilution  of  the  Patient's 
Serum. — There  are  several  methods  of  collecting  blood 
from  the  patient,  which  will  be  given  in  the  order 
preferred. 

(1)  By  means  of  Wright's  capsule:  A  piece  of 
%-inch  glass  tubing  is  drawn  out  to  a  fine  capillary 
stem  at  each  end,  leaving  a  portion  with  the  original 
calibre  about  2  inches  in  length.  One  of  the  capillary 
ends  of  the  tube  should  be  bent  (see  Fig.  22,  B),  The 


Fig.  4. — Capillary  teat  pipette  for  removal  of  serum  from  clotted  specimen  of  blood. 

patient's  blood  is  drawn  into  the  curved  end  of  the  cap- 
sule by  capillar}^  attraction  from  a  puncture  of  the 
finger  or  lobe  of  the  ear.  When  the  tube  is  almost  full, 
the  ends  can  be  sealed  in  a  small  flame.  After  the 
blood  has  clotted  and  the  serum  separated,  a  file  mark 
is  made  at  a  convenient  level  on  the  tube,  which  is  then 
broken,  and  the  clear  serum  drawn  off  by  means  of  a 
capillary  pipette  (Fig.  4).  Any  desired  dilution  of 
the  serum  can  now  readily  be  made  by  taking  one  drop 
of  serum  from  the  pipette,  and  the  required  number 
of  drops  of  distilled  water  with  the  same  pipette.  The 
dilutions  usually  employed  in  performing  the  Widal 


AGGLUTININS  75 

reaction  are  1  in  40  and  1  in  80.  The  dilutions  are 
conveniently  made  in  small  test-tubes  or  watch-glasses. 
For  the  1  in  40  dilution,  one  drop  of  serum  is  placed 
in  the  test-tube  or  watch-glass  by  means  of  the  pipette, 
and  19  drops  of  distilled  water  are  then  added  with 
the  same  pipette.  This  makes  a  1  in  20  dilution  of  the 
serum,  a  platinum  loopful  of  which  added  to  a  loopful 
of  bouillon  typhoid  culture  gives  the  desired  1  in  40 
dilution.  Similarly  for  the  1  in  80  dilution,  one  drop 
of  serum  and  39  drops  of  water  are  mixed  in  a  test- 
tube,  making  a  dilution  of  1  in  40.  A  loopful  of  this 
together  with  a  loopful  of  typhoid  culture  gives  the  1 
in  80  dilution.  This  is  the  most  reliable  method  of 
preparing  the  patient's  serum,  as  it  is  free  from  red 
cells,  and  the  dilution  is  accurate. 

(2)  By  means  of  a  leucocyte  counting  pipette: 
The  blood  is  di-awn  up  into  the  leucocyte  counting 
pipette  of  the  Thoma-Zeiss  apparatus,  as  far  as  the 
0.5  mark,  just  as  for  a  leucoc}i;ic  count.  Instead  of 
the  acetic  acid  used  in  the  latter  procedure,  however, 
the  tube  is  filled  up  to  the  11  mark  with  distilled  water, 
thus  immediately  giving  a  dilution  of  1  in  20.  This 
is  the  most  rapid  method  of  preparing  the  patient's 
blood,  and  works  very  satisfactorily  in  hospital  prac- 
tice, where  the  test  is  usually  carried  out  immediately 
lafter  collection  of  the  blood.  The  red  cells  interfere 
very  little,  if  any,  with  the  reaction. 


76  APPLIED  IMMUNOLOGY 

(3)  Employment  of  dried  blood:  Two  or  three 
drops  of  patient's  blood  are  collected  upon  a  clean 
glass  slide  or  non-absorbent  paper  and  allowed  to  dry 
there.  In  making  the  dilution,  enough  water  is  added 
to  the  dried  blood  to  replace  that  lost  in  drying,  and 
the  clot  well  broken  up  in  it  by  means  of  a  platinum 
needle.  The  dilution  can  then  be  made  by  drops  from 
a  capillary  pipette  as  described  above.  This  method 
is  useful  in  cases  where  the  necessary  apparatus  for 
either  of  the  other  two  methods  is  not  at  hand,  and 
where  the  blood  has  to  be  sent  for  examination  a  dis- 
tance. While  the  matter  of  dilution  by  this  means  is 
largely  guesswork,  reliable  results  can  be  obtained 
from  it,  though  if  possible  it  is  better  to  use  one  of  the 
other  methods  above  described. 

{b)  Preparation  of  the  Culture  of  Typhoid 
Bacilli. — A  stock  agar  culture  of  typhoid  bacilli  should 
be  kept  at  hand,  and  should  be  renewed  by  transfer- 
ring to  a  fresh  agar  tube  every  three  or  four  months. 
For  use  in  the  test  a  bouillon  culture  is  made  from  this. 
The  bouillon  culture  should  be  24  to  48  hours  old, 
grown  at  room  temperature.  In  such  a  culture  the 
typhoid  bacilli  are  found  to  be  present  in  sufficient 
numbers,  of  large  size,  and  very  motile.  A  satisfac- 
tory bouillon  culture  can  be  kept  constantly  on  hand 
by  inoculating  a  fresh  tube  with  the  preceding  one 
every  48  hours.    If  the  bacilli  die  out  or  prove  other- 


AGGLUTININS  77 

wise  unsatisfactory,  a  bouillon  tube  can  be  inoculated 
from  the  original  agar  culture.  Before  using  a  cul- 
ture for  the  Widal  test,  the  organisms  should  always 
be  examined  for  motility. 

(c)  Technic  of  the  Widal  Reaction. — The  micro- 
scopic test  is  usually  carried  out  with  a  depressed 
hanging-drop  slide.  The  edge  of  the  depression  is 
ringed  with  vaseline.  Upon  a  clean  cover-slip  are 
placed  one  platinum  loopful  of  the  bouillon  culture  of 
typhoid  bacilli,  and  one  loopful  of  previously  diluted 
1  in  20  patient's  serum,  giving  a  dilution  of  1  in  40. 
The  slide  is  pressed  down  upon  the  cover-glass,  and 
quickly  inverted  so  that  the  hanging-drop  remains  in 
the  centre  of  the  depression.  A  similar  slide  is  pre- 
pared with  a  dilution  of  1  in  80,  and  a  third  slide  with 
culture  only,  to  servx  as  a  control  for  the  motility  of 
the  typhoid  bacilli.  Each  slide  is  marked  with  the 
dilution  and  time  of  beginning  the  test  by  means  of  a 
grease  pencil.^  Each  slide  in  turn  is  now  placed  under 
the  microscope,  the  high-power  dry  objective  (%  in.) 
being  used.  The  typhoid  bacilli  will  be  seen  to  move 
in  all  directions  through  the  field.  In  from  %  to  1  hour 
in  the  case  of  a  positive  reaction,  all  the  typhoid  bacilli 

*  We  have  found  that  plain  glass  slides  can  be  used  with  equal  satis- 
faction, as  reconunended  by  Stitt.  A  ring  of  vaseline  is  made  on  the 
centre  of  the  slide,  which  is  then  gently  allowed  to  rest  on  the  cover-glass 
without  any  other  pressure  than  its  own  weight.  Sufficient  space  is  thus 
preserved  to  allow  of  motility  of  the  organisms. 


78  APPLIED  IMMUNOLOGY 

in  dilution  1  in  40  and  probably  also  in  1  in  80  will 
have  lost  their  motility,  and  most  of  them  will  be 
collected  together  in  clumps  throughout  the  field 
(Fig,  5) .  In  the  control  slide,  containing  no  serum,  the 
organisms  at  the  end  of  this  time  \W11  still  retain  their 
motility,  and  there  will  be  no  clumping  ( Fig.  6 ) .  In 
case  of  a  negative  reaction,  the  bacteria  in  all  the  slides 
remain  motile.  If  the  slide  in  which  a  positive  reaction 


Fig.  5. — Widal's  test,   positive   (aggluti-  Fig.  6. — Widal'e  test,  negative, 

nation  or  clumping). 

is  taking  place  be  observed  from  time  to  time,  at  first 
a  few  of  the  bacteria  will  be  seen  to  lose  their  motility, 
and  one  will  become  attached  to  another,  giving  a  V- 
or  Y-shaped  appearance.  These  groups  will  gradu- 
ally be  added  to  by  other  bacilli  until  clmnps  are 
formed  and  all  motility  is  lost.  Various  grades  of  in- 
tensity of  reaction  are  seen,  ranging  from  formation 
of  a  few  clumps  and  many  organisms  retaining  their 


AGGLUTININS  79 

motility  to  complete  clumping  and  loss  of  motility  of 
all.  The  degree  of  clumping  should  be  reported  as 
a  partial  reaction,  which,  although  suggestive,  is  not 
necessarily  specific  for  typhoid  fever.  Various  grades 
of  intensity  are  sometimes  seen  in  the  different  dilu- 
tions. A  positive  reaction  with  1  in  40  dilution  may 
practically  always  be  regarded  as  specific  for  typhoid 
fever,  though  occasionally  blood  of  patients  infected 
with  B,  paratyphosus  A  or  B  may  give  a  positive  re- 
action in  this  dilution  with  typhoid  bacilli.  A  positive 
result  with  1  in  80  dilution  renders  the  diagnosis 
doubly  sure. 

B.        MACROSCOPIC   METHOD  OF   PERFORMING  THE  WIDAL 
REACTION 

For  this  method  small  test-tubes,  such  as  are  em- 
ployed in  the  Wassermann  reaction,  are  used.  Serum 
in  different  dilutions  is  placed  in  the  tubes  by  means  of 
small  pipettes,  and  quantities  of  bouillon  culture  of 
typhoid  bacilli  are  then  added.  A  control  tube  con- 
sisting of  the  culture  alone  is  also  used.  At  the  end  of 
12  to  24  hours  at  37°  C,  in  the  case  of  a  positive  re- 
action, the  bacilli  become  clumped  and  fall  as  a  sedi- 
ment to  the  bottom  of  the  tube,  leaving  a  clear  fluid 
above.  In  the  case  of  a  negative  reaction  and  in  the 
control  tube,  the  fluid  remains  uniformly  cloudy.  The 
reaction  can  be  studied  best  by  means  of  a  reading 
glass.    In  this  method  killed  cultures  of  the  typhoid 


80  APPLIED  IMMUNOLOGY 

bacilli  may  be  employed,  and  have  the  advantage  of 
keeping  in  good  condition  for  some  time.    With  ordi- 
nary laboratory  facilities,  however,  the  microscopic 
method  is  preferable. 
Occurrence  and  Significance  of  the  Widal  Reaction 

The  Widal  reaction  is  usually  not  seen  in  typhoid 
fever  until  the  second  week  of  the  disease,  as  some 
time  is  required  for  the  production  of  the  specific  ag- 
glutinins in  the  patient's  serum,  upon  which  the  test 
depends.  The  reaction  usually  persists  for  several 
months  after  recovery  from  typhoid  fever,  and  per- 
sons have  been  known  to  react  positively  for  years 
after  the  infection.  Artificial  immunization  with  ty- 
phoid vaccine  also  gives  rise  to  a  positive  reaction 
which  persists  for  some  time.  These  facts  should  be 
remembered  in  weighing  the  results  of  the  test  per- 
formed in  suspected  cases  of  the  disease.  The  diag- 
nosis of  typhoid  fever  should  not  rest  upon  the  agglu- 
tination reaction  alone.  Other  symptoms  must  be  con- 
sidered along  with  the  laboratory  test.  The  reaction 
may  fail  to  appear  in  a  small  percentage  of  cases  of 
undoubted  typhoid  fever,  and  may  be  positive  occa- 
sionally in  other  conditions,  particularly  in  jaundice. 
Probably  the  range  of  error  on  either  side  does  not  ex- 
ceed 3  per  cent.,  so  that  in  the  Widal  reaction  we  have 
one  of  the  most  important  cardinal  signs  of  typhoid 
fever. 


AGGLUTININS  81 

Other  Applications  of  the  Agglutination  Phenomenon 

For  the  identification  of  certain  species  of  bacteria, 
the  agglutination  reaction  is  of  the  greatest  value. 
For  this  purpose,  agglutinins  are  artificially  produced 
by  injection  of  animals,  usually  rabbits,  with  cultui^es 
of  the  various  organisms  whose  presence  is  suspected 
in  given  cases.  Thus  we  have  conditions  reversed  from 
those  obtaining  in  the  Widal  reaction  for  typhoid 
fever,  the  serum  being  the  known  factor,  and  the  or- 
ganism the  unknown.  For  example,  we  have  isolated 
an  organism  that  we  strongly  suspect  to  be  the  ty- 
phoid bacillus.  If  we  find  that  a  culture  of  this  or- 
ganism is  agglutinated  by  the  greatly  diluted  serum 
from  an  animal  previously  immunized  to  typhoid 
bacilli,  and  is  not  agglutinated  by  similar  dilutions  of 
sera  from  animals  immunized  to  other  organisms,  such 
as  B.  jparatyphosus,  dy sentence,  etc.,  our  suspicion  is 
converted  into  certainty.  This  method  is  of  great 
value,  especially  in  the  detection  of  typhoid  bacilli  in 
milk  and  other  fluids,  as  well  as  in  other  procedures. 


X 

PRECIPITINS 

SIGNIFICANCE  AND  APPLICATION  OF  PRECIPITINS— 
TECHNIC  OF  REACTION— SPECIFIC  IDENTIFICATION  OF 
BLOOD  AND  OTHER  PROTEINS 

When  a  protein  is  brought  in  relation,  in  vitro, 
with  blood-serum  of  an  animal  which  has  been  immu- 
nized against  that  particular  protein,  the  protein  is 
thrown  out  of  solution,  the  fluid  becomes  cloudy,  and  a 
sediment  finally  settles  to  the  bottom  of  the  tube.  This 
is  known  as  the  precipitation  phenomenon,  and  is  due 
to  the  presence  in  the  blood-serum  of  substances  known 
as  precipitins.  Precipitins  are  antibodies  formed  in 
the  blood  in  response  to  injection  of  unorganized  pro- 
tein material.  They  are  antibodies  of  the  second  order 
of  Ehrlich,  and  are  analogous  to  agglutinins,  which 
act  upon  organized  proteins  or  bacteria. 

The  precipitation  phenomenon  is  made  use  of  in  the 
identification  of  various  proteins.  Each  species  of  ani- 
mal has  a  specific  protein,  which  can  be  detected  by  the 
precipitation  reaction.  Practically,  the  test  has  been 
made  use  of  chiefly  in  ( I )  detection  of  adulteration  of 
meat  products  such  as  sausages,  etc. ;  and  ( II )  in  the 
medicolegal  identification  of  blood. 

(I)  Detection  of  Adulteration  of  Meat  Products 

This  test  can  be  applied  to  determine  whether 
sausages  represented  as  being  made  purely  of  beef  or 

82 


PRECIPITINS  83 

pork,  contain  horse  meat.  Preparation  for  the  test 
consists  in  the  immunization  of  animals,  of  which  rab- 
bits are  the  most  suitable,  by  injections  of  extracts 
made  from  the  various  meats  to  be  tested  for,  as  horse, 
beef,  pork,  etc.,  each  of  which  has  a  specific  protein. 
The  immune  serum,  on  being  brought  in  contact  with 
an  extract  of  the  particular  meat  whose  protein  is 
specific  for  it,  will  bring  about  precipitation. 

(II)  Medicolegal  Identification  of  Blood 

This  test  occasionally  assumes  the  utmost  impor- 
tance when  the  question  arises  in  murder  cases  as  to 
whether  blood  stains  are  human  or  not.  The  chemical 
tests  for  blood  do  not  distinguish  one  species  from 
another,  while  microscopical  examination  of  dried 
blood  is  unsatisfactory.  Dried  blood  in  very  small 
quantities,  however,  will  respond  to  the  precipitation 
reaction,  and  by  this  means  we  can  differentiate 
human  blood  from  that  of  the  lower  animals.  Uhlen- 
huth,  Wassermann,  and  Schiiltz  were  the  first  to  make 
use  of  the  precipitation  test  in  this  connection. 

The  Precipitin  Reaction 

The  technic  of  the  reaction  is  divided  into  two 
parts. 

( a )  Production  of  the  Precipitating  Serum, — For 
this  purpose  rabbits  are  employed.    Three  or  four  in- 


84 


APPLIED  IMMUNOLOGY 


travenous  injections  of  human  blood-serum  are  made 
into  the  rabbit  in  quantities  of  1-2  c.c.  every  five  or 
six  days.  The  strength  of  the  antihuman  serum  thus 
formed  may  be  tested  after  three  or  four  injections. 
This  is  obtained  by  piercing  a  vein  in  the  ear  of  the 
rabbit,  collecting  a  few  cubic  centimetres  of  blood,  al- 
lowing the  clear  serum  to  separate,  and  titrating  it 
with  different  dilutions  of  human  serum.  The  dilu- 
tions are  to  be  made  with  normal  salt  solution.  The 
antiserum  may  be  regarded  as  of  sufficient  strength 
when  0.1  c.c  will  produce  distinct  cloudiness  when 
mixed  with  1  c.c.  of  human  serum  in  1-1000  dilution 
after  incubation  for  five  minutes  at  37°  C.  The  anti- 
serum, moreover,  must  give  no  cloudiness  with  plain 
salt  solution,  nor  with  the  serum  of  any  other  animal 
in  1-200  dilution  after  half  an  hour  at  37°  C.  The  fol- 
lowing table  illustrates  the  titration  of  a  suitable  anti- 
human  serum : 


Antihuman 
rabbit  serum 


Human 
serum  1  c.c. 


Controls 


Result  after  incuba- 
tion for  y^  hour  at 
37°  C. 


0.1 

0.1 
0.1 
0.1 
0.1 
0.1 

0.1 


1-10 

l-IOO 

1-1000 

1-5000 

1-10000 


1  c.c.  normal  salt 
solution 

1  c.c.  normal 
guinea-pig  ser- 
um (1-200) 


Marked  turbidity- 
Marked  turbidity- 
Marked  turbidity- 
Turbidity 
Slight  turbidity- 
Clear 

Clear 


PRECIPITINS 


85 


(b)  Identification  of  Blood. — An  extract  of  the 
blood  clot  is  made  with  normal  salt  solution.  This  is 
then  filtered  until  absolutely  clear,  and  diluted  until 
we  have  approximately  a  1-1000  solution  of  the  serum. 
The  latter  point  can  be  determined  by  boiling  a  por- 
tion of  the  solution,  as  a  1-1000  dilution  will  then  show 
a  slight  haziness,  while  greater  dilutions  remain  clear. 

One-tenth  c.c.  of  the  antihuman  rabbit's  serum  is 
then  mixed  with  1  c.c.  of  the  suspected  serum  thus  pre- 
pared, and  incubated  for  half  an  hour  at  37°  C.  In 
another  tube  0.1  c.c.  of  normal  rabbit  serum  is  placed 
with  1  c.c.  of  the  suspected  serum.  In  a  third  tube  is 
placed  0.1  c.c.  of  antihuman  rabbit  serum  and  1  c.c. 
of  normal  salt  solution.  The  results  in  the  case  of  a 
positive  reaction  are  shown  in  the  following  table : 


Antihuman 
rabbit 
serum 

Suspected 
blood 

Normal 

salt 
solution 

Normal 
rabbit 
serum 

Result.  S?"  C. 

After 
5  minutes 

After 
half  hour 

1 

2 
3 

c.c. 

0.1 
0.1 

1    (1-1000) 

i" (i-ioob) 

c.c. 

1 

0^1 

Turbidity 

Negative 
Negative 

Marked 
turbidity 
Negative 
Negative 

It  has  not  been  found  possible  to  employ  this  test 
for  differentiation  of  the  blood  of  various  races  of 
human  beings,  such  as  negro  from  Caucasian,  etc. 

The  same  test  can  be  used  for  the  blood  of  various 
lower  animals  by  preparing  suitable  antisera.  Closely 
related  species,  however,  such  as  hens  and  pigeons,  are 
not  differentiated  by  it. 


XI 

LYSINS 

BACTERIOLYSINS  AND  CYTOLYSINS  (HEMOLYSINS) 

AiviONG  specific  antibodies  of  the  third  order  of 
Ehrlich  we  find  hacteriolysins,  which  are  substances 
acquired  by  the  blood-serum  of  an  animal  that  is  im- 
munized to  specific  bacteria.  Bacteriolysins  have  the 
property  of  dissolving  the  particular  bacteria  for 
which  they  are  specific.  Analogous  substances  are 
likewise  formed  against  body-cells  known  as  cytoly- 
sins.  Cytolysins  that  act  upon  red  blood-corpuscles 
are  called  hcemolysins.  In  order  for  the  phenomenon 
of  bacteriolysis  to  take  place,  in  addition  to  the  specific 
bacteriolysin  (also  known  as  amboceptor) ,  the  pres- 
ence of  a  second  or  non-specific  substance  is  necessary. 
This  substance  is  known  as  a  complement,  and  is  pres- 
ent in  varying  amount  in  the  blood-serum  of  all  ani- 
mals whether  immunized  or  not.  The  same  comple- 
ment, being  non-specific,  may  take  part  in  any  bac- 
teriolytic or  cytolytic  reaction.  Amboceptors  are  said 
to  be  thermostable,  Le.,  they  are  not  destroyed  by  heat- 
ing to  56°  C,  while  complement  is  thermolahile ,  and 
is  destroyed  at  a  temperature  of  5Q°  C.  The  non- 
specific element  or  complement  can  therefore  be  re- 

86 


LYSINS  87 

moved  from  a  serum  by  heating  the  latter  to  this  tem- 
perature, rendering  the  serum  inactive  although  it  con- 
tains the  specific  element  or  amboceptor.  The  serum 
can  be  made  active  again  by  adding  more  complement 
in  the  form  of  fresh  unheated  normal  serum.  This 
discovery  that  two  elements,  one  specific  and  the  other 
non-specific,  are  necessary  for  bacteriolysis,  was  made 
by  Bordet. 

Pfeiffer's  Phenomenon. — Our  knowledge  of  bac- 
teriolysins  is  dependent  largely  upon  the  following 
experiment  carried  out  by  PfeifFer.  He  injected  a 
suspension  of  cholera  spirilla  into  the  peritoneal  cavity 
of  guinea-pigs  that  had  been  previously  immunized  to 
cholera  spirilla,  and  also  into  non-immunized  guinea- 
pigs.  In  the  case  of  the  non-immunized  animals  the 
bacteria  increased  in  number  until  they  destroyed  the 
animals.  In  the  case  of  the  previously  immunized 
guinea-pigs,  examination  of  the  peritoneal  fluid  from 
time  to  time  showed  that  the  bacteria  quickly  died,  be- 
came clumped  together  in  granules,  became  broken  up, 
and  finally  were  dissolved.  Practically,  this  phenome- 
non may  be  made  use  of  in  the  identification  both  of 
bacteria  and  of  specific  immune  sera.  It  is  especially 
of  value  in  the  identification  of  cholera  spirilla.  For 
this  purpose,  guinea-pigs  previously  immunized  to  chol- 
era are  injected  intraperitoneally  with  the  suspected 
organisms,  and  the  peritoneal  fluid  examined  from 


88  APPLIED  IMMUNOLOGY 

time  to  time  for  the  occurrence  of  bacteriolysis.  Pas- 
sively immunized  animals  may  also  be  employed  for 
the  observation  of  Pf eiff  er's  phenomenon.  That  is,  the 
reaction  will  occur  on  injection  of  a  mixture  of  cholera 
immune  serum  and  a  culture  of  cholera  spirilla  into  the 
guinea-pig.  This  phenomenon  can  also  be  observed  in 
vitro,  by  mixing  immune  serum  and  bacteria  in  a  test- 
tube,  and  incubating  at  37°  C.  for  some  time. 

What  has  been  said  of  bacteriolysins  regarding  the 
manner  in  which  they  operate,  applies  likewise  to 
cytolysins  and  haemolysins.  Thus,  for  example,  if  a 
specific  immune  serum,  which  has  been  prepared  by 
injecting  the  blood-corpuscles  of  a  sheep  into  the  peri- 
toneal cavity  of  a  rabbit,  is  mixed  with  the  corpuscles 
of  the  sheep,  and  incubated  at  37°  C,  the  sheep's  cor- 
puscles will  after  a  short  time  be  dissolved  (haemoly- 
sis). If  the  rabbit's  serum  be  previously  heated  to 
56°  C.  for  half  an  hour,  haemolysis  will  not  take  place 
because  the  complement  or  non-specific  factor  in  the 
reaction  will  have  been  destroyed.  The  immune  serum, 
though  it  still  retains  its  specific  factor  or  hsemolysin 
(amboceptor),  has  been  inactivated.  The  serum  can 
be  re-activated  by  the  addition  of  fresh  normal  serum 
of  any  animal,  thus  supplying  complement,  after 
which  the  hsemolytic  action  will  take  place  as  before. 

In  the  reaction  by  antibodies  belonging  to  the 
third  order  of  Ehrlich,  whether  they  be  bacteriolytic, 


LYSINS  89 

cytolytic,  or  hasmolytic,  the  substances  acted  upon 
(bacteria,  blood-corpuscles,  etc.)  are  known  as  anti- 
gens; the  specific  antibody  (bacteriolysin,  cytolysin, 
hsemolysin)  is  spoken  of  as  amboceptor;  while  the  non- 
specific factor  in  the  reaction  is  known  as  complement. 
Various  diagnostic  tests  of  the  greatest  practical 
importance  have  this  reaction  as  a  basis,  and  will  re- 
ceive consideration  in  the  following  chapter. 


XII 

FIXATION  OF  COMPLEMENT 

PRINCIPLES  OF  THE  REACTION— BORDET-GENGOU  PHE- 
NOMENON—WASSERMANN-NEISSER-BRUCK  MODIFICA- 
TION—TECHNIC  OF  THE  WASSERMANN  REACTION  IN 
THE  DIAGNOSIS  OF  SYPHILIS— MODIFICATIONS  OF  THE 
WASSERMANN  REACTION— HECHT-WEINBERG  MODIFI- 
CATION—CLINICAL APPLICATION  OF  THE  WASSER- 
MANN REACTION— EFFECTS  OF  TREATMENT  ON  THE 
WASSERMANN  REACTION 

In  the  foregoing  chapter  we  have  seen  that  bac- 
teriolysis, cytolysis,  and  hsemolysis  take  place  by 
means  of  a  specific  substance  known  as  amboceptor, 
and  a  non-specific  substance  called  complement.  This 
imion  of  antigen,  amboceptor,  and  complement  is 
spoken  of  as  fixation  or  ahsoi'ption  of  complement. 
The  exact  nature  of  the  reaction  is  not  known.  In 
order  that  the  complement-fixation  reaction  be  under- 
stood in  its  application  to  specific  diagnosis,  it  is  neces- 
sary to  explain  first  in  some  detail  the  process  of 
hgemolysis.  When  red  blood-corpuscles  remain  in  sus- 
pension in  a  fluid,  the  fluid  has  an  opaque  red  color. 
If  haemolysis  occurs,  the  haemoglobin  leaves  the  cor- 
puscles, the  fluid  becomes  transparent,  and  has  a  deep 
red  color.  If  no  haemolysis  occurs,  the  corpuscles 
gradually  fall  to  the  bottom  of  the  tube,  leaving  clear, 

colorless  fluid  above.    Haemolysis  can  occur  in  a  non- 
90 


FIXATION  OF  COMPLEMENT  91 

specific  manner,  i.e,,  without  amboceptor  and  comple- 
ment, through  several  agencies.  Thus,  if  the  cor- 
puscles be  placed  in  plain  water,  haemolysis  will  occur, 
and  likewise  through  the  addition  of  various  substances, 
such  as  snake  venom,  tetanus  toxin,  etc.  Specific 
haemolysis  differs  from  non-specific  in  that  it  will 
take  place  in  an  isotonic  fluid  such  as  physiologic  salt 
solution. 

In  haemolysis  there  is  no  actual  solution  of  the  cell. 
The  process  consists  rather  in  a  disturbance  of  the  os- 
motic equilibrium  between  the  cell  contents  and  the 
surrounding  medium.  The  haemolytic  amboceptor 
combines  with  the  stroma  of  the  cell,  increases  the 
permeability  of  the  latter,  setting  free  the  haemoglobin 
into  the  surrounding  medium.  Bacteriolysis  is  de- 
pendent upon  a  similar  mechanism,  and  differs  from 
haemolysis  in  that  it  is  practically  invisible  to  the  naked 
eye. 

The  blood  of  most  animals  usually  contains  a  cer- 
tain amount  of  natural  haemolysin  for  the  red  cor- 
puscles of  other  animals.  Thus,  human  blood-serum 
possesses  a  considerable  natural  haemolytic  power  for 
sheep's  corpuscles.  This  normal  haemolytic  power  can 
be  greatly  increased  artificially  by  injection  of  the 
blood-corpuscles  of  one  animal  into  another,  so  that 
very  high  dilutions  of  the  second  animal's  serum  will 
haemolyze  the  corpuscles  of  the  first,  in  the  presence  of 


92  APPLIED  IMMUNOLOGY 

suitable  amounts  of  complement.  In  experimental 
and  diagnostic  work,  the  rabbit  is  the  animal  usually 
employed  for  the  production  of  specific  hemolytic 
serum.  By  three  or  four  intraperitoneal  or  intraven- 
ous injections  of  suitable  quantities  of  washed  sheep's 
corpuscles  into  the  rabbit  at  intervals  of  3  or  4  days, 
the  rabbit's  serum  will  become  so  highly  immunized 
that  it  will,  in  1-1000  or  greater  dilution,  hsemolyze 
an  equal  quantity  of  5  per  cent,  suspension  of  sheep's 
corpuscles  in  the  presence  of  a  sufficient  amount  of 
complement.  It  has  been  found  that  complement  is 
present  in  varying  amounts  in  the  blood-serum  of  dif- 
ferent animals,  but  that  the  serum  of  the  guinea-pig 
is  most  constant  in  this  respect.  So  in  order  to  insure 
the  presence  of  a  definite  amount  of  complement,  the 
immune  hsemolytic  serum  (rabbit)  is  heated  at  56°  C. 
to  destroy  the  complement  present,  and  a  definite 
amount  of  fresh  guinea-pig  serum  afterwards  added. 
The  technic  of  the  preparation  and  titration  of  specific 
hsemolytic  serum  will  be  given  under  the  description  of 
the  Wassermann  reaction. 

The  Bordet-Gengou  Phenomenon 

Bordet  and  Gengou  were  the  first  to  employ  the 
complement-fixation  reaction  in  the  diagnosis  of  spe- 
cific infections.  Since  the  phenomena  of  bacteriolysis 
and  of  haemolysis  each  depend  upon  the  presence  and 


FIXATION  OF  COMPLEMENT  93 

fixation  of  a  common  non-specific  substance  termed 
complement,  these  observers  conceived  the  idea  of 
combining  the  two  phenomena  as  a  means  of  diagnosis. 
The  components  of  the  first-mentioned  reaction,  con- 
sisting of  the  bacterial  suspension  or  antigen,  the  spe- 
cific antibacterial  serum  or  amboceptor,  and  comple- 
vient,  together  form  the  bacterial  system;  the  second 
part  is  known  as  the  hcemolytic  system,  consisting  of 
blood-corpuscles  (antigen),  specific  hsemolytic  serum 
(amboceptor) ,  and  complement.  Bordet  and  Gengou 
took  a  suspension  of  cholera  spirilla  as  antigen  and 
mixed  it  with  a  certain  quantity  of  cholera-immune 
serum  made  by  injecting  a  rabbit  with  cholera  spirilla, 
together  with  a  definite  amount  of  fresh  guinea-pig 
serum  as  complement.  This  mixture  was  incubated  at 
37 ""  C.  for  one  hour.  At  the  end  of  this  time  it  was  pre- 
sumed that  the  interaction  between  antigen  and  ambo- 
ceptor had  taken  place  with  fixation  or  absorption  of 
complement.  But  since  the  reaction  was  not  percepti- 
ble to  the  eye,  a  further  procedure  became  necessary  to 
show  the  result.  For  this  purpose,  a  suspension  of 
washed  rabbit's  blood-corpuscles  and  a  quantity  of 
hsemolytic  serum  for  the  rabbit's  corpuscles  were 
added,  and  the  whole  incubated  for  another  hour.  At 
the  end  of  this  time,  it  was  found  that  no  haemolysis  of 
the  rabbit's  corpuscles  had  taken  place,  because  the 
complement  had  been  fixed  during  the  first  incubation 


94  APPLIED  IMMUNOLOGY 

by  the  bacterial  system,  none  being  left  free  to  act  in 
connection  with  the  hsemolytic  system. 

In  a  second  experiment,  exactly  the  same  compon- 
ents were  used,  except  that  the  cholera-immune  serum 
was  replaced  by  normal  serum.  Hence,  complement 
was  not  fixed  by  the  bacterial  system,  but  remained 
free  to  become  a  part  of  the  hsemolytic  system,  and 
haemolysis  occurred. 

The  first  or  positive  and  the  second  or  negative  re- 
actions may  be  graphically  illustrated  thus : 

1.  Positive  Result 

Suspension  of  cholera  spirilla  Haemolytic  serum         No 

^  v^  haemolysis 

X  _^^,,^  Complement 

Cholera-inamune  serum '^''''''^  Red  corpuscles 

2.  Negative 

Suspension  of  cholera  spirilla  Haemolytic  serum 

Complement  ^  Haemolysis 

^^  T 

Normal  serum  Red  corpuscles 

The  specific  complement-fixation  reaction  may  be 
applied  to  the  diagnosis  of  several  diseases.  By  test- 
ing an  unknown  serum  with  a  known  bacterial  anti- 
gen, it  is  thus  possible  to  determine  whether  or  not  the 
individual  furnishing  the  serum  is  infected  by  the  par- 
ticular organism  from  which  the  antigen  is  made.  For 
some  time  after  Bordet  and  Gengou's  announcement, 
the  reaction  had  chiefly  a  scientific  rather  than  a  prac- 
tical interest.  The  first  great  advance  toward  render- 
ing the  method  of  practical  importance  was  the  dis- 


FIXATION  OF  COMPLEMENT  95 

covery  by  Wassermann  and  Bruck  that  extracts  fur- 
nished by  lysis  of  the  bacteria  will  sei^'e  as  antigens 
for  the  complement -fixation  reaction  equally  as  well 
as,  if  not  better  than,  suspensions  of  the  bacteria  them- 
selves. This  fact  was  applied  by  the  workers  men- 
tioned in  the  diagnosis  of  typhoid  fever,  tuberculosis, 
meningitis,  etc. 

The  Wassermann  Reaction 

A  further  step  was  now  taken  by  Wassermann, 
Neisser,  and  Bruck.  They  recognized  what  an  enor- 
mous value  the  reaction  would  have  if  it  could  be  ap- 
plied to  syphilis,  the  causative  organism  of  which  had 
not  been  artificially  cultivated  up  to  that  time  (1906) . 
So  these  observers  conceived  the  idea  of  employing  as 
antigen,  extracts  of  organs  kno\\Ti  to  be  rich  in  tre- 
ponemata  (spirochastae).  From  this  the  now  uni- 
versally used  and  important  "  Wassermann  reaction  " 
had  its  origin.  In  their  first  experiments  Wassermann, 
Neisser,  and  Bruck  made  water}^  extracts  from  syphi- 
litic fetal  liver,  in  which  Treponemata  pallida  are  very 
abundant.  Using  these  extracts  they  obtained  fixa- 
tion of  complement  with  the  sermn  of  syphilitic  in- 
dividuals, and  from  their  results  concluded  that  the  re- 
action was  specific,  just  as  that  obtained  in  the  case  of 
typhoid  bacillus  extract  and  typhoid  serum.  But  it 
was  then  found  that  alcoholic  extracts  of  syphilitic  tis- 


96  APPLIED  IMMUNOLOGY 

sues  would  give  the  reaction  in  syphilis  with  equally 
good  results.  Since  it  is  the  lipoid  material  in  tissue 
that  is  dissolved  by  alcohol,  it  followed  that  this  lipoid 
material  must  contain  the  substance  that  is  antigenic 
for  syphilitic  serum. 

It  was  then  found  by  Marie,  Levaditi,  and  Land- 
steiner  that  complement-fixation  with  syphilitic  serum 
occurred  when  alcoholic  extracts  of  normal  tissues,  such 
as  liver,  human  heart,  or  guinea-pig  heart,  were  used 
as  antigen,  and  that  the  results  of  reactions  with  these 
extracts  were  as  reliable  as  those  where  extracts  of 
syphilitic  tissues  were  used.  These  observations 
showed  that  the  complement-fixation  reaction  in  syph- 
ilis is  not  entirely  a  specific  antigen-antibody  reaction 
as  in  the  case  of  those  occurring  between  bacterial  anti- 
gens and  antibodies,  but  this  fact  in  no  way  lessened 
the  practical  value  of  the  test  in  the  diagnosis  of 
syphilis. 

It  has  still  further  been  shown  that  extracts  pre- 
pared from  lecithin,  cholesterin,  etc.,  can  be  used  as 
antigens  and  some  workers  restrict  themselves  to  these 
artificial  products.  The  various  forms  of  antigen  in 
use  at  the  present  time  account  to  a  great  extent  for 
the  divergence  of  results  in  some  cases  when  serum 
from  the  same  patient  is  examined  by  different 
workers.  This  has  a  tendency  to  throw  doubt  on  the 
value  of  the  reaction  in  the  minds  of  skeptical  clini- 


FIXATION  OF  COMPLEMENT  97 

cians.  A  great  deal  of  work  is  being  done  upon  the 
antigen  question,  and  it  is  hoped  that  in  the  near  fu- 
ture some  definite  standard  will  be  adopted  and  uni- 
versally employed.  We  believe,  in  the  light  of  pres- 
ent knowledge,  that  in  the  Wassermann  reaction  there 
is  probably  a  specific  factor  as  well  as  a  non-specific, 
certainly  that  the  organisms  of  syphilis  cause  an  over- 
production of  the  special  lipoid  antigenic  substance  in 
the  tissues,  and  that  the  most  reliable  results  will  be 
obtained  by  using  an  alcoholic  extract  of  syphilitic 
tissue,  in  which  this  substance  is  found  in  greatest 
abundance.  We  adhere  to  this  view  as  the  result  of 
the  use  of  syphilitic  liver-extract  in  several  thousand 
tests,  in  which  our  results  have  differed  in  no  material 
way  from  those  of  reliable  workers. 

We  recently  reported  (J.  A.  M.  A,,  Jan.  31, 
1914)  the  results  of  a  trial  of  some  cholesterinized  ex- 
tracts of  guinea-pig  heart  and  human  heart.  These 
extracts  are  made  by  adding  cholesterin  to  the  ordi- 
nary alcoholic  extract  of  normal  heart  in  the  propor- 
tion of  0.4  per  cent.  The  cholesterinized  guinea-pig 
heart-extract  was  used  by  us  along  with  our  antigen  of 
alcoholic  extract  of  syphilitic  liver  in  the  routine  ex- 
amination of  100  cases,  the  cholesterinized  human 
heai-t-extract  being  employed  in  thirty-three  cases. 

Careful  preliminary  titration  of  the  antigens  was 
carried  out  before  doing  the  tests,  and  at  no  time  was 

7 


98  APPLIED  IMMUNOLOGY 

more  than  one-third  of  the  anticomplementary  dose 
used.  The  comparative  results  of  tests  with  syphilitic 
liver-extract  and  cholesterinized  guinea-pig  heart-ex- 
tract may  be  divided  into  four  groups  as  follows : 

1.  In  twenty-two  cases  the  results  were  positive 
with  both  antigens,  with  little  or  no  diiFerence  in  de- 
gree of  reaction. 

2.  In  eight  cases  the  syphilitic  liver-extract  gave  a 
weakly  positive  reaction,  while  the  cholesterinized  ex- 
tract gave  a  stronger  reaction. 

3.  In  thirty-six  cases  both  antigens  gave  a  negative 
result. 

4.  In  thirty-four  cases  the  syphilitic  liver-extract 
gave  a  negative  reaction,  while  the  cholesterinized 
heart-extract  gave  a  weakly  positive  or  medium  posi- 
tive result.  This  is  naturally  the  most  important 
group  from  the  clinical  point  of  view.  Nine  of  these 
thirty-four  cases  were  treated  cases  of  syphilis  ex- 
hibiting no  symptoms,  showing  that  it  is  more  difficult 
to  render  the  serum  negative  to  the  cholesterinized 
heart-extract  by  anti-syphilitic  treatment.  The  re- 
maining twenty-five  of  this  group  were  cases  sub- 
mitted for  diagnosis  or  which  had  no  signs  or  history 
of  syphilis.  When  one  antigen  reacted  more  strongly 
than  the  other,  it  was  always  the  cholesterinized  heart- 
extract.  In  no  case  did  the  syphilitic  liver-extract  give 
a  stronger  reaction  than  the  cholesterinized  extract. 


FIXATION  OF  COMPLEMENT  99 

In  the  comparative  tests  with  the  cholesterinized 
extract  of  human  heart,  of  thirty-three  cases  there 
were  six  in  the  first  group,  that  is,  equally  positive 
with  the  two  extracts;  four  in  the  second  group,  in 
which  the  cholesterinized  human  heart-extract  gave  a 
distinctly  stronger  reaction  than  the  syphilitic  liver 
antigen;  sixteen  in  the  third  group,  in  which  both 
antigens  gave  negative  results;  seven  in  the  fourth 
group,  which  were  negative  with  the  syphilitic  anti- 
gen but  positive  with  the  cholesterinized  heart-extract. 

In  the  first  series,  therefore,  the  results  with  the 
two  antigens  disagreed  in  42  out  of  100  cases,  and  in 
the  second  series  in  11  out  of  33  cases.  We  see  from 
these  results  that  the  cholesterinized  extracts  appar- 
ently give  a  more  "  delicate  "  reaction  than  the  ex- 
tract of  syphilitic  liver,  but  we  also  find  that  many 
weakly  positive  results  are  obtained  by  them  in  non- 
syphilitic  cases.  This  fact  alone  is  quite  sufficient,  in 
our  opinion,  to  offset  any  advantage  in  delicacy  of  the 
reaction  obtained  with  the  use  of  cholesterinized  ex- 
tracts. We  feel  that  just  as  high  a  percentage  of 
positive  results  is  obtained  in  known  or  clinically  ap- 
parent syphilitics  with  the  syphilitic  liver-extract  as  is 
obtained  with  the  cholesterinized  antigens,  and  the 
former  do  not  give  positive  results  in  non-syphilitic 
cases.  The  success  in  detecting  mild  degrees  of  syphi- 
litic infection  is  doubtless  due  in  part  to  the  use  of  one 


100  APPLIED  IMMUNOLOGY 

properly  standardized  unit  of  complement  and  hsemo- 
lytic  amboceptor,  as  suggested  to  us  by  Laird,  instead 
of  double  or  triple  units  as  employed  by  many 
workers.  We  must  conclude  that  with  cholesterinized 
antigens,  varying  degrees  of  inhibition  of  hsemolysis 
may  be  obtained  in  serums  from  many  conditions 
other  than  syphilis,  and  in  normal  persons.  While  ex- 
perimental investigations  are  to  be  highly  commended, 
we  therefore  regard  the  employment  of  these  artificial 
antigens  for  routine  clinical  use  at  the  present  stage  of 
our  knowledge,  instead  of  being  an  advance  in  sero- 
logic technic,  rather  as  a  distinct  step  backward. 

Schamberg,  Kolmer,  and  others  report  that  they 
obtained  positive  Wassermann  reactions,  using  the 
cholesterinized  antigens  in  over  28  per  cent,  of  twenty- 
two  cases  of  psoriasis,  in  a  great  many  of  which  syphi- 
lis could  almost  certainly  be  excluded,  thus  providing 
evidence  that  weak  reactions  with  these  antigens  do 
not  necessarily  mean  syphilis,  and  that  a  diagnosis  of 
syphilis  cannot  be  based  on  weak  and  medium  inhibi- 
tions when  they  are  employed.  We  hold  that  weakly 
positive  reactions  with  syphilitic  liver-extract  mean 
nothing  but  syphilis.  Even  though  it  were  true  that 
the  cholesterinized  antigens  give  a  more  "  delicate  " 
reaction  and  may  furnish  positive  results  in  cases  of 
syphilis  that  are  negative  to  the  syphilitic  liver-ex- 
tract, it  is  a  very  much  less  serious  error  to  overlook  an 


FIXATION  OF  COMPLEMENT  101 

occasional  case  of  syphilis  than  to  saddle  a  diagnosis 
of  the  disease  with  all  it  entails  on  a  patient  who  does 
not  have  the  disease. 

Considerable  harm  is  being  done  at  present  by  the 
use  of  unreliable  non-specific  or  artificial  extracts,  in 
two  ways: 

1.  The  marked  discrepancies  between  the  results 
of  the  Wassermann  test  and  the  clinical  findings  in 
many  cases  are  causing  skeptical  clinicians  to  lose  con- 
fidence in  the  value  of  the  reaction,  and  thus  they  are 
being  deprived  of  an  important  diagnostic  and  thera- 
peutic aid, 

2.  A  great  many  unfortunate  persons  are  being 
treated  for  syphilis  who  have  not  and  never  had  syphi- 
lis, as  the  result  of  weakly  positive  and  doubtful  re- 
ports of  workers  using  these  antigens. 

Since  Noguchi,  in  1911,  first  cultivated  the  Tre- 
ponema pallidum  in  a  pure  state,  much  work  has 
been  done  by  Noguchi,  Kolmer,  and  others  with 
antigens  made  from  the  treponemata  themselves.  The 
results  have  been  disappointing  for  diagnosis  of  pri- 
mary and  early  secondary  syphilis,  as  only  a  relatively 
small  proportion  of  these  cases  gives  positive  reactions 
when  the  spirochsetal  antigens  are  employed.  But  the 
positive  reactions  obtained  in  late  secondary  and  ter- 
tiary cases  do  prove  that  there  is  a  specific  fixation  of 
complement  occurring  when  lipoidal  substances  are 


102 


APPLIED  IMMUNOLOGY 


brought  in  relation  with  syphihtic  serum,  but  as  yet  we 
are  in  the  dark  in  regard  to  the  true  nature  of  the 
phenomenon. 

Several  factors  other  than  the  antigen  are  impor- 
tant in  obtaining  reliable  results  with  the  Wassermann 
reaction;  the  most  essential  of  which  is  the  titration 
and  use  of  minimal  amounts  of  complement. 


Technic  of  the  Wassermann  Reaction 

The  following  is  a  list  of  the  apparatus,  animals, 
etc.,  required,  and  has  been  made  as  complete  as 
possible : 


Electric  centrifuge. 
Several    15    c.c.    graduated   centri- 
fuge tubes. 
Several  1   c.c.  pipettes,  graduated 

in  tenths  of  a  c.c. 
Several  1.2  c.c.  pipettes,  graduated 

in  hundredths  of  a  c.c. 
Two  10  c.c.  pipettes,  graduated  in 

tenths  of  a  c.c. 
Capillary    pipettes,     prepared     by 

drawing  out  3/8  in.  glass  tubing. 
Rubber      nipples      for      capillary 

pipettes. 
100  1  c.c.  glass  ampoules. 
100  small  test-tubes,   about  5   c.c. 

capacity. 
Several  flasks,  capacity  500  c.c. 
Two  flasks,  100  c.c.  capacity. 
Two   graduated   100  c.c.  cylinders. 
Several    Petri    dishes. 


Normal  salt  solution  (0.85  per 
cent.). 

Sodium  citrate  solution  (1  per 
cent,  in  normal  salt  solution). 

Wire  racks  for  holding  small  test- 
tubes. 

Platinum  loop  with  glass  handle. 

Scissors,  scalpel,  dissecting  forceps. 

2  c.c.  all  glass  hypodermic  syringe 
with  small  needle. 

Wax  pencil. 

95  per  cent,  alcohol,  ether,  xylol, 
glycerin. 

Water-bath. 

Centigrade  thermometer. 

Incubator. 

Bunsen  burner. 

Refrigerator. 

Sheep,  rabbits,  guinea-pigs. 


All   glass-ware    and   solutions   mentioned   above 
must  be  sterilized  before  using. 


FIXATION  OF  COMPLEIVIENT  103 

HEMOLYTIC  System 

We  employ  the  anti-sheep  hasmolytic  system,  i.e., 
washed  sheep's  red  blood-corpuscles  and  anti-sheep 
hemolytic  rabbit  serum.  As  the  hsemolytic  ambocep- 
tor requires  some  time  in  preparation,  its  production 
will  be  described  first.  This  anti-sheep  heemolytic 
serum — which  hereafter  for  the  sake  of  brevity  will 
be  termed  amboceptor — is  prepared  by  injecting 
quantities  of  washed  sheep's  red  blood-corpuscles  into 
a  rabbit  until  in  the  presence  of  a  suitable  amount  of 
complement  the  rabbit's  serum  in  high  dilution  will 
dissolve  corpuscles  from  the  sheep  when  properly  in- 
cubated in  vitro. 

Collection  of  Sheep's  Blood. — The  collection  of 
blood  and  preparation  of  suspension  or  red  corpuscles 
as  described  below  is  the  same  for  the  test  itself  as  for 
injection  into  the  rabbit.  A  sheep  may  be  kept  on  the 
premises,  or  the  blood  may  be  obtained  from  a  nearby 
slaughter-house.  The  latter  if  available  saves  a  good 
deal  of  time.  The  sheep's  blood  can  be  drawn  from  the 
internal  jugular  vein  by  plunging  a  needle  of  large 
lumen  into  the  vein  and  allowing  the  blood  to  fall  into 
a  flask  containing  pieces  of  glass  or  wire.  The  blood 
is  defibrinated  and  thus  prevented  from  clotting  by 
shaking  the  flask  immediately  after  collection  of  the 
blood.  For  the  reaction,  a  5  per  cent,  suspension  of 
sheep's  corpuscles  is  employed.    Whole  blood  consists 


104  APPLIED  IMMUNOLOGY 

approximately  of  one-half  serum  and  one-half  cor- 
puscles. To  make  up  a  5  per  cent,  suspension  there- 
fore, 1  c.c.  of  the  defibrinated  blood  is  placed  in  a 
graduated  centrifuge  tube  by  means  of  a  1  c.c.  pipette, 
and  the  contents  of  the  tube  brought  up  to  10  c.c.  by 
the  addition  of  normal  salt  solution.  This  tube  then 
will  contain  0.5  c.c.  or  5  per  cent,  of  sheep's  corpuscles. 
Several  tubes  can  be  prepared  in  the  same  way,  to 
furnish  sufficient  suspension  of  corpuscles  for  the  test. 
A  convenient  method  used  by  us  for  obtaining 
blood  from  the  sheep  is  as  follows :  In  several  gradu- 
ated centrifuge  tubes  are  placed  9  c.c.  of  1  per  cent, 
sodium  citrate  solution  in  normal  saline.  The  sheep's 
ear  is  washed  off  with  alcohol,  and  one  of  the  large 
veins  severed  with  a  scalpel  (Fig.  7) .  One  c.c.  of  the 
blood  is  now  allowed  to  drop  into  each  of  the  tubes, 
the  sodium  citrate  preventing  it  from  clotting.  This 
gives  a  5  per  cent,  suspension  of  sheep's  corpuscles. 
It  is  necessary  that  all  serum  be  removed  from  the  sus- 
pension, in  order  to  avoid  anaphylactic  symptoms  if 
for  injection  into  the  rabbit,  and  to  remove  all 
complement  if  the  corpuscles  are  to  be  used  in  the  test 
itself.  This  is  accomplished  by  centrifuging  the  tubes 
for  5  or  6  minutes  in  an  electric  centrifuge,  at  the  end 
of  which  time  the  corpuscles  wiU  be  found  at  the 
bottom.  The  volume  of  the  corpuscles  will  vary  ac- 
cording to  the  length  of  time  used  in  centrifuging,  the 


FIXATION  OF  COMPLEMENT 


105 


longer  the  time  the  more  closely  will  they  be  packed. 
From  this  it  is  easily  seen  that  the  method  used  of  tak- 
ing a  definite  amount  of  whole  blood  in  the  beginning 
and  from  that  calculating  the  dilution  required  is  more 
accurate  than  drawing  out  a  quantity  of  the  centri- 
fuged  corpuscles  and  making  the  dilution  from  them, 


Fig.  7. — Method  of  obtaining  blood  from  sheep's  ear. 

which  is  the  method  commonly  employed.  The  clear 
fluid  above  the  corpuscles  is  poured  off,  and  replaced 
with  normal  salt  solution,  the  tubes  shaken,  and  again 
centrifuged.  This  washing  and  centrifuging  is  re- 
peated three  or  four  times,  in  order  to  remove  all 
serum.  If  the  corpuscle  suspension  is  to  be  used  in 
the  test  itself,  the  fluid  in  each  tube  is  finally  brought 


106  APPLIED  IMMUNOLOGY 

up  to  its  original  volume  with  normal  salt  solution, 
making  a  5  per  cent,  suspension.  Practically,  a  larger 
amount  of  suspension  can  be  prepared  in  the  same 
number  of  centrifuge  tubes,  by  originally  placing  say 
3  c.c.  of  whole  blood  in  each,  washing  and  centrifug- 
ing  three  or  four  times,  and  after  the  final  washing 
bringing  the  volume  up  to  30  c.c.  For  injection  into 
the  rabbit,  a  10  per  cent,  suspension  of  the  corpuscles 
is  more  suitable  than  a  5  per  cent,  suspension,  so  after 
the  final  washing  the  fluid  in  a  tube  containing  origi- 
nally 1  c.c.  of  whole  blood  or  0.5  c.c.  of  corpuscles  is 
brought  up  to  a  total  volume  of  only  5  c.c. 

Immunization  of  the  Rabbit  to  Sheep's  Corpuscles, 
— The  suspension  of  sheep's  corpuscles  prepared  as 
described  i^ove  may  be  injected  into  the  rabbit  either 
intraperitoneally  or  intravenously.  Since  much  larger 
amounts  of  suspension  and  a  longer  time  for  immu- 
nization are  required  by  the  former  method,  we  have 
discarded  it  entirely  in  favor  of  the  intravenous 
method.  The  latter  is  just  as  simple,  and  if 
proper  precautions  against  infection  and  injection 
of  air  be  taken,  is  quite  safe.  For  the  first  injection 
about  1  c.c.  of  the  10  per  cent,  corpuscle  suspension  is 
drawn  up  into  an  all-glass  hypodermic  syringe  of  2 
c.c.  capacity  and  fitted  with  the  usual  fine  needle;  the 
smaller  the  needle  the  better.  The  marginal  vein  of 
the  rabbit's  ear  is  made  prominent  by  wiping  it  with 


FIXATION  OF  COMPLEMENT 


107 


xylol,  and  the  suspension  slowly  injected  into  it 
through  the  skin  in  the  direction  of  the  circulation 
(Fig.  8).  Usually,  to  obtain  a  sufficiently  powerful 
h^emolytic  amboceptor,  three  injections  at  intervals  of 
3  or  4  days  are  required.    The  second  injection  should 


Fig.  8. — Showing  method  of  intravenous  injection  or  immunization  of  rabbit  with 
10  per  cent,  suspension  of  sheep's  blood-corpuaclea  for  the  production  of  haemolytio 
amboceptor. 

consist  of  1.5  c.c.  and  the  third  of  2  c.c.  of  the  sus- 
pension. Three  or  4  days  after  the  third  injection, 
about  2  c.c.  of  the  rabbit's  blood  is  drawn  from  the  ear 
by  puncturing  a  vein,  and  the  serum  tested  for  its 
hsemolytic  power  against  sheep's  corpuscles.    Thus  in 


108  APPLIED  IMMUNOLOGY 

ten  days,  or  two  weeks  at  the  most,  we  are  able  to  pre- 
pare a  powerful  hgemolytic  amboceptor,  which  re- 
quires four  or  five  weeks  by  the  intraperitoneal 
method. 

Titration  of  Hcemolytic  Amboceptor, — For  use  in 
the  Wassermann  test  we  aim  to  produce  a  hsemolytic 
amboceptor  of  such  strength  that  1  c.c.  of  not  less  than 
a  1-2000  dilution  of  rabbit's  serum  will  hasmolyze  1 
c.e.  of  a  5  per  cent,  suspension  of  sheep's  corpuscles 
in  the  presence  of  0.1  c.c.  of  fresh  guinea-pig  serum 
used  as  complement.  Usually,  by  the  intravenous 
method  described,  the  rabbit  can  be  immunized  so  that 
its  serum  in  1-3000  dilution  will  fulfil  these  re- 
quirements. To  determine  the  strength  of  the  hsemo- 
lytic serum,  three  or  four  days  after  the  third  injection 
of  the  rabbit,  about  2  c.c.  of  blood  are  drawn  into  a 
small  test-tube  from  a  vein  of  the  rabbit's  ear.  When 
the  blood  has  clotted  and  the  clear  serum  separated, 
the  latter  is  drawn  oiF  and  the  natural  complement  in 
it  destroyed  by  heating  the  serum  in  a  water-bath  for 
half  an  hour  at  56°  C.  This  deprivation  of  comple- 
ment is  known  as  inactivation.  The  complement  used 
in  titrating  the  hsemolytic  amboceptor  and  in  the 
Wassermann  test  itself  is  furnished  by  serum  of  the 
guinea-pig,  as  it  has  been  found  that  this  animal  has 
an  almost  constant  amount  of  complement  present  in 
its  blood.  To  obtain  complement,  a  full-grown  guinea- 


/^      --,  -R 


Fig.  9. — Demonstrating   method    of    obtaining    complement   by   bleeding  to  death    an 
anaesthetized  guinea-pig. 


FIXATION  OF  COMPLEMENT  109 

pig  is  etherized  by  placing  its  head  in  a  glass  contain- 
ing gauze  soaked  with  ether,  and  before  death  occurs, 
the  blood-vessels  of  the  neck  are  severed  and  the  blood 
collected  in  a  Petri  dish  (Fig.  9) .  More  blood  will  be 
obtained  if  drawn  before  respiratory  and  cardiac 
movements  cease,  and  from  10  to  15  c.c.  should  be  col- 
lected in  this  manner.  The  blood  is  allowed  to  coagu- 
late, and  the  clear  serum  to  separate,  which  it  does  in 
about  two  hours.  The  serum  is  then  drawn  off  with  a 
capillary  pipette  and  diluted  in  the  proportion  of  one 
part  in  ten  with  0.85  per  cent,  salt  solution,  i.e.,  to  1 
part  of  serum  9  parts  of  salt  solution  are  added.  The 
complemental  property  of  serum  is  soon  lost,  and  the 
guinea-pig  serum  should  therefore  be  used  on  the  day 
it  is  obtained,  or  not  later  than  the  following  day,  if 
kept  on  ice  over  night. 

The  different  reagents  having  been  prepared  as 
above  described,  we  are  now  ready  for  the  titration. 
The  rabbit's  serum,  after  having  been  inactivated,  is 
made  up  in  the  following  dilutions:  1-500,  1-1000, 
1-1500,  1-2000,  1-3000,  1-4000,  and  1-5000,  in  nor- 
mal salt  solution.  In  a  series  of  test-tubes  1  c.c.  of 
each  of  these  dilutions  is  placed,  together  with  1  c.c.  of 
a  5  per  cent,  suspension  of  sheep's  corpuscles  and  0.1 
c.c.  of  the  1-10  dilution  of  guinea-pig  complement, 
with  sufficient  salt  solution  to  bring  the  total  volume 
up  to  4  c.c.    After  their  contents  have  been  thoroughly 


110 


APPLIED  IMMUNOLOGY 


mixed  by  inversion,  the  tubes  are  placed  in  the  incu- 
bator for  one  hour  at  37°  C  After  this  time,  if  the 
amboceptor  is  sufficiently  powerful  for  practical  use, 
the  corpuscles  in  the  tubes  containing  1-500,  1-1000, 
1-1500,  and  1-2000  dilutions  of  rabbit's  serum  should 
be  completely  hasmolyzed.  Those  in  the  tubes  con- 
taining higher  dilutions  generally  show  partial  hsemol- 
ysis.  The  following  table  illustrates  the  titration  of 
haemolytic  amboceptor: 


Amboceptor 


dilution 

1-500 

1-1000 

1-1500 

1-2000 

1-3000 


1       1-4000 
1       1-5000 


Comple- 
ment 


0.1 
0.1 
0.1 
0.1 
0.1 

0.1 
0.1 


5  per  cent,  suspen- 

sion of  sheep's 

cor- 

puscles 

p 

ex. 

o' 

1 

13 

1 

t-* 

1 

1 

P 

1 

CO 

1 

o 

1 

P 

Result 


Complete  hsemolysis 
Complete  liEemolysis 
Complete  haemolysis 
Complete  hsemolysis 
Almost    complete 

hsemolysis 
Partial  hsemolysis 
Partial  hsemolysis 


If  the  1-2000  tube  does  not  show  complete  haemol- 
ysis, the  rabbit  must  be  given  another  injection  of 
sheep's  corpuscles  in  order  to  raise  the  hsemolytic 
power  of  the  serum.  If  the  serum  is  found  to  be  of 
sufficient  hsemolytic  strength,  the  rabbit  should  be 
etherized  and  bled  to  death  from  the  carotid  artery 
'  without  delay,  as  the  serum  in  the  living  animal  will 
lose  its  hsemolytic  power  after  a  time.  The  rabbit's 
blood  is  collected  in  Petri  dishes,  and  set  aside  on  ice 
imtil  the  clear  serum  separates.     This  is  drawn  oif 


FIXATION  OF  COMPLE^IENT  111 

with  a  capillary  pipette,  inactivated  by  heating  for 
half  an  hour  at  56°  C,  and  stored  for  future  use  in 
1  c.c.  glass  ampoules.  Amboceptor,  when  collected  in 
this  way  under  aseptic  conditions,  will  retain  its  ha^mo- 
lytic  power  for  several  months.  We  have  found  that 
the  serum  is  less  likely  to  deteriorate  or  become  con- 
taminated when  mixed  with  an  equal  quantity  of 
glycerin,  as  suggested  by  Much.  The  glycerin  in  no 
way  interferes  with  the  Wassermann  test.  We  also 
prefer  to  store  the  glycerinized  serum  in  short  lengths 
of  glass  tubing  with  sealed  ends,  as  suggested  to  us  by 
Laird,  because  a  larger  amount  of  air  must  be  en- 
closed in  the  ampoules,  with  consequent  greater  risk  of 
contamination.  In  the  Wassermann  test  itself  we  em- 
ploy 1  c.c.  of  a  1-1000  dilution  of  hemolytic  ambo- 
ceptor that  gives  a  titre  of  at  least  1-2000.  We  avoid 
having  to  open  a  fresh  tube  of  amboceptor  and  thus 
wasting  a  great  deal,  by  making  a  1-100  dilution  from 
one  tube,  placing  it  in  sl  sterile  flask,  and  making  the 
1-1000  dilution  from  this  for  several  days. 

Syphilitic  System 

Antigen. — For  reasons  given  above,  we  prefer  to 
use  as  antigen  an  alcoholic  extract  made  from  the 
liver  of  a  syphilitic  foetus  that  has  been  found  on  micro- 
scopic examination  to  be  rich  in  treponemata.  The 
method  usually  employed  in  preparing  such  an  anti- 


112  APPLIED  IMMUNOLOGY 

gen  is  as  follows :  One  part  of  the  tissue  is  minced  and 
mixed  with  four  parts  of  96  per  cent,  alcohol.  The 
antigenic  substances  are  then  extracted  by  shaking  the 
mixture  in  a  machine  for  24  hours,  when  the  fluid  is 
filtered  or  allowed  to  stand  until  the  sediment  falls  to 
the  bottom  of  the  vessel.  The  clear  fluid  is  then 
pipetted  off*  and  placed  in  dark  bottles,  to  be  used  as 
required.  This  extract  must  be  tested  for  antigenic 
power  against  a  number  of  known  syphilitic  and 
known  non-syphilitic  sera,  and  must  give  positive  re- 
sults with  the  former  and  negative  with  the  latter  in 
order  to  be  fit  for  use.  Furthermore,  the  most  suit- 
able dose  must  be  ascertained.  When  present  in  ex- 
cessive amount,  the  extract  may  cause  fixation  of  com- 
plement alone  in  the  absence  of  syphilitic  serum.  The 
optimal  dose  of  antigen  is  that  which  gives  complete 
complement-fixation  (or  inhibition  of  hsemolysis) 
with  a  syphilitic  serum  and  three  times  which  is  re- 
quired to  produce  partial  fixation  without  syphilitic 
serum.  Briefly,  the  optimal  dose  of  antigen  should 
not  be  greater  than  one-third  the  anti-complementary 
dose.  This  dose  is  determined  by  performing  the 
Wassermann  test  on  a  syphilitic  serum  using  several 
tubes  containing  the  serum  and  various  amounts  of  an- 
tigen. Other  series  of  tubes  are  set  up  containing  2,  3, 
and  4  times  these  amounts  of  antigen  but  without 
syphilitic  serum,  to  determine  the  anti-complementary 


FIXATION  OF  COMPLEMENT 


lis 


dose  of  antigen.  This  procedure  will  be  understood 
more  clearly  after  the  Wassermann  test  itself  has  been 
described  (Fig.  10).  In  our  own  work  we  rely  en- 
tirely upon  an  imported  German  extract  made  as  de- 
scribed above,  which  we  have  found  to  retain  its  anti- 


Fig.  10. — Titration  of  antigen. 

Front  row:  Increasing  quantities  of  antigen  tested  with  0.1  c.c.  of  known  syphilitic 
serum  to  ascertain  optimal  dose. 

Back  row:  Determination  of  anti-complementary  dose  of  antigen.  Each  tube  con- 
tains 3  times  the  amount  of  antigen  present  in  corresponding  tube  of  front  row,  but 
no  serum. 

genie  power  almost  indefinitely,  and  which  has  very 
slight  anti-complementary  action.  By  its  use  we  avoid 
the  rather  tedious  preparation  and  titration  of  anti- 
gen, except,  of  course,  that  we  make  a  test  of  anti- 
complementary power  with  each  new  bottle,  and  use 
8 


114 


APPLIED  IMMUNOLOGY 


kno\vn  positive  and  negative  controls  every  day  the 
test  is  performed. 

Collection  and  Preparation  of  the  Patient's  Serum, 
— The  patient's  blood  to  be  tested  may  be  drawn  from 
a  vein  at  the  bend  of  the  elbow  with  a  hypodermic 
syringe  and  immediately  transferred  to  a  small  test- 
tube.  There  are  several  other  methods  in  use,  as  by 
means  of  Keidel's  vacuum  ampoule  (Fig.  11),  cup- 


FiG.  11. — Showing  method  of  collecting  blood  from  vein  of  arm  with  Keidel's 
vacuum  ampoule.  Observe  the  tourniquet  of  rubber  dam  secured  with  the  easily  de- 
tached, tucked-under  loop. 

ping,  etc.  At  least  2  c.c.  of  blood  should  be  obtained. 
The  method  employed  by  us  is  as  follows :  The  ring- 
finger  is  wiped  off  with  alcohol,  allowed  to  dry,  and 
then  given  a  deep  puncture  at  the  radial  extremity  of 
the  distal  phalanx  about  a  quarter  of  an  inch  below 
the  angle  of  the  nail  with  a  large  Hagedom  needle. 
The  blood  is  then  squeezed  out  into  a  small  test-tube 
by  intermittently  compressing  the  radial  side  of  the 


FIXATION  OF  COMPLEIMENT 


115 


patient's  finger  with  the  tip  of  the  little  finger  of  the 
right  hand  holding  the  test-tube,  the  thumb  and  index- 
finger  of  the  left  hand  holding  and  constricting  the 
circulation  on  the  other  three  sides  of  the  patient's 
finger  (see  Fig.  12) .    With  a  little  practice,  sufficient 


Fig.  12. — Showing  authors'  method  of  obtaining  blood  for  complement-fixation 
reactions.  Note  the  little  finger  of  the  right  hand  holding  the  test-tube,  free  to  exert 
intermittent  pressure  over  the  radial  aspect  of  the  patient's  punctured  finger,  held  and 
constricted  on  three  sides  by  the  enveloping  thumb  and  index-finger  of  the  left  hand. 

blood  can  thus  be  obtained  in  a  minute  or  two,  and  the 
method  does  not  entail  any  formidable  preparation 
and  obviates  the  possibility  of  phlebitis  incident  to 
faulty  venous  puncture.  In  very  young  children,  suf- 
ficient blood  can  quickly  be  obtained  by  puncturing 


116  APPLIED  IMMUNOLOGY 

the  lieeL  The  tube  containing  the  blood  thus  collected 
should  be  plugged  with  cotton  and  if  possible  be  kept 
in  the  refrigerator  until  required  for  the  test.  It 
will  keep  well  for  four  or  five  days  in  this  way,  but 
should  be  tested  as  early  as  possible  after  removal 
from  the  patient.  If  blood  is  to  be  carried  or  sent  any 
distance,  a  cork  or  other  impervious  stopper  should  be 
put  in  the  tube,  as  inversion  would  cause  the  serum  to 
be  absorbed  by  a  cotton  stopper.  The  blood  if  kept 
too  long,  or  in  too  high  temperature,  may  heemolyze, 
and  is  then  unfit  for  use,  though  slight  hsemolysis  does 
not  interfere  with  the  test.  Consequently  if  the  serum 
is  to  be  shipped  from  a  distance  or  stored  miusually 
long,  the  serum  had  best  be  separated  from  the  clot. 

We  now  arrive  at  the  procedures  to  be  carried  out 
on  the  day  of  the  test.  They  will  be  given,  as  far  as 
possible,  in  the  order  in  which  they  may  be  carried  out 
in  the  shortest  possible  time. 

Pi^eparation  of  Comjylement. — The  guinea-pig  is 
first  of  all  bled  to  death  in  the  manner  previously  de- 
scribed, so  that  its  serum  will  have  separated  by  the 
time  it  is  required.  One  guinea-pig  as  a  rule  will  fur- 
nish sufficient  complement  for  about  40  cases  and 
their  controls.  The  serum  is  made  into  a  1  in  10  dilu- 
tion with  normal  saline  solution  for  convenience  in 
handling  small  quantities. 

Preparation  of  the  Suspension  of  Sheep's  Cor- 


FIXATION  OF  COMPLEMENT  117 

pnscles. — Sixty  c.c.  of  a  5  per  cent,  suspension  of 
sheep's  corpuscles  will  usually  be  found  ample  for 
about  twenty  cases.  This  suspension  is  made  as  de- 
scribed under  the  hsemolytic  system.  The  washed 
corpuscles  will  keep  on  ice  without  deterioration  for 
about  three  days,  after  which  spontaneous  haemolysis 
usually  begins. 

Titration  of  Complement, — In  performing  the 
Wassermann  test  it  is  essential  that  we  know  the 
smallest  amount  of  complement  that  is  necessary  to 
produce  haemolysis  in  the  heemolytic  system.  There- 
fore before  coming  to  the  test  proper  we  must  each 
day,  first  of  all,  find  out  the  smallest  amount  of  guinea- 
pig  serum  in  the  presence  of  which  1  c.c.  of  a  1-1000 
dilution  of  amboceptor  will  heemolyze  1  c.c.  of  a  5 
per  cent,  suspension  of  sheep's  corpuscles.  This 
amount  is  known  as  1  unit  of  complement,  and  may 
vary  somewhat  in  the  serum  of  different  guinea-pigs. 
The  employment  of  not  more  than  one  unit  of  comple- 
ment in  the  reaction  is  of  great  importance,  since  an 
excess  might  furnish  enough  to  produce  complete 
haemolysis  and  thus  give  a  negative  result  when  some 
of  the  complement  employed  has  been  previously 
fixed  by  syphilitic  serum  and  antigen.  Again,  the 
use  of  less  than  one  unit  of  complement  would  give 
a  positive  reaction  even  in  the  absence  of  syphilitic 
serum,  as  haemolysis  would  then  be  incomplete.    The 


118  APPLIED  IMMUNOLOGY 

majority  of  workers  employ  excessive  doses  of  comple- 
ment and  double  or  treble  the  smallest  amount  of 
amboceptor  necessary  to  produce  complete  hemolysis 
of  the  corpuscles.  The  only  possible  reason  for  the 
use  of  such  excessive  doses  of  the  two  reagents  is 
to  overcome  the  effect  of  fixation  of  a  slight  amount 
of  complement,  non-specifically,  by  the  patient's 
serum  and  the  antigen  alone,  and  to  replace  the  com- 
plemental  power  lost  in  the  incubation.  This  empiri- 
cal method  is  responsible  in  part  for  negative  results 
in  positive  cases.  By  the  method  in  use  by  us,  we 
are  enabled  to  accurately  allow  sufficient  complement 
for  the  overcoming  of  any  non-specific  fixation  by 
serum  and  antigen  alone,  and  also  to  make  up  for 
the  complement  lost  during  incubation,  and  at  the 
same  time  use  in  the  test  proper  not  more  than  one 
hemolytic  unit.  Previous  to  titration  of  comple- 
ment with  hemolytic  amboceptor  and  corpuscles,  the 
complement  is  first  incubated  with  one  dose  of  antigen 
and  0.1  c.c.  of  a  mixture  of  several  non-syphilitic 
sera.  This  is  done  for  the  reason  that  a  slight  amount 
of  complement  is  fixed  under  these  conditions  by  the 
serum  and  antigen,  and  we  are  thus  sure  of  allowing 
for  this  slight  non-specific  fixation  in  the  test  itself. 
Different  sera  present  variations  in  the  amount  of 
complement  they  are  capable  of  absorbing  in  this 


FIXATION  OF  COMPLEMENT 


110 


manner,  but  these  variations  are  usually  so  slight 
that  for  practical  purposes  they  can  be  ignored, 
particularly  if  a  pooled  serum  from  several  non- 
syphilitic  cases  be  used.  The  technic  of  titration 
of  complement  is  carried  out  as  follows:  Into  each 
of  a  series  of  six  tubes  is  placed  0.2,  0.3,  0.4,  0.5,  0.6, 


Fig.  13. — Titration  of  complement.  With  the  complement  in  each  tube  are  placed 
0.1  c.c.  of  non-syphilitic  serum  and  1  dose  of  antigen.  The  tubes  contain  gradually 
increasing  amounts  of  complement,  ranging  from  0.2  c.c.  (dilution  1-10)  to  0.8  c.c. 

0.7  and  0.8  c.c.  of  1-10  dilution  of  guinea-pig  serum, 
together  with  0.1  c.c.  of  pooled  non-syphilitic  serum 
previously  inactivated  by  heating  at  56°  C.  for  half  an 
hour,  and  the  dose  of  antigen  used  in  the  test  (Fig. 
13) .  Sufficient  salt  solution  is  then  added  to  bring  the 
volume  up  to  2  c.c.    The  tubes  are  shaken  and  incu- 


120 


APPLIED  IMMUNOLOGY 


bated  for  one  hour  at  37°  C,  after  which  1  c.c.  of 
1-1000  hsemolytic  amboceptor  and  1  c.c.  of  5  per  cent, 
suspension  of  sheep's  corpuscles  are  added  to  each 
tube.  After  a  further  incubation  of  one  hour,  the  de- 
gree of  haemolysis  that  has  taken  place  in  the  different 
tubes  is  noted.  Usually  complete  hsemolysis  has  oc- 
curred in  all  the  tubes  containing  0.5  c.c.  and  more  of 
diluted  complement.  The  0.5  c.c.  tube  is  therefore 
said  to  contain  one  unit  of  complement  plus  the  small 
extra  amount  allowed  for  non-specific  inhibition  due 
to  the  presence  of  patient's  serum  and  antigen.  This 
is  the  amount  of  complement  to  be  used  in  the  test 
proper  on  that  particular  day.  The  table  given  below 
will  illustrate  the  titration  of  complement : 


Pooled 

Antigen 
1-7 

Comple- 

Ambo- 

Sheep's 

^ 

O 

normal 
serum 

ment 
1-10 

O 

g- 

ceptor 
1-1000 

cles  5 
percent. 

C 
cr 
P 

Result 

o 

h 

c.c. 

c.c. 

c.c. 

p 

c  c. 

c.c. 

E3 

1 

0.1 

0.2 

0.2 

H- 

1 

1 

1— ' 

Slight  haemolysis 

2 

0.1 

0.2 

0.3 

1 

1 

•-1 

Partial  hsemolysis 

3 

0.1 

0.2 

0.4 

!  P 

1    en- 

1 

1 

Almost  complete 

d- 

haemolysis 

4 

0.1 

0.2 

0.5 

^ 

1 

1 

ii 

Complete  hemolysis 

5 

0.1 

0.2 

0.6 

p 

1 

1 

o 

P 

Complete  haemolysis 

6 

0.1 

0.2 

0.7 

1 

1 

Complete  haemolysis 

By  making  two  parallel  experiments,  it  is  readily 
shown  that  where  the  complement  has  been  previously 
subjected  to  incubation  with  inactivated  normal  serum 
and  antigen,  in  the  same  doses  as  are  used  in  the  test 
itself,  a  larger  amount  is  required  for  hsemolysis  than 


FIXATION  OF  COMPLEMENT 


121 


where  complement  after  being  incubated  for  one  hour 
alone  is  added  to  the  amboceptor  and  sheep's  cor- 
puscles. In  a  second  series  of  tubes  prepared  in  this 
way  the  results  would  be : 


Comple- 

Ambocep- 

Sheep's 

t3 

ment 

tor 

corpuscles 

2 

1-10 

1-1000 

5  per  cent. 

COST. 

c.c. 

c.c. 

c.c. 

^® 

1 

0.2 

1 

op 

2 

0.3 

1 

3 
4 

04 
0.5 

1 
1 

?l 

5 

0.6 

1 

p  ! 

Result 


Partial  haemolysis 
Almost  complete  hsemolysis 
Coiri'plete  hceinolysis 
Complete  haemolysis 
Complete  haemolysis 


By  this  means  we  arrive  at  the  exact  amount  of 
guinea-pig  serum  that  represents  one  unit  of  comple- 
ment. This  will  be  of  practical  value  later  as  seen  in 
quantitative  tests. 

Patient's  Serum. — The  serimi  of  the  patient's 
blood  will  usually  separate  spontaneously  from  the 
clot  and  can  be  drawn  off  with  a  capillary  pipette.  If 
not,  the  clot  can  be  broken  up  well  with  a  platinum 
loop  and  the  serum  separated  by  centrifuging  the 
tube.  The  clear  serum  can  then  be  readily  drawn  off. 
The  patient's  serum  is  heated  in  a  water-bath  at  5^""  C. 
for  half  an  hour  to  destroy  the  natural  complement 
present  (inactivation) .  This  can  be  carried  out  while 
the  complement  is  being  titrated.  At  the  same  time  a 
known  syphilitic  and  a  known  non-syphilitic  ^erum 
should  be  inactivated  to  be  used  in  the  test  as  controls. 


122  APPLIED  IMMUNOLOGY 

Performance  of  the  Wassermann  Test  Proper 

The  performance  of  the  test  on  one  unknown 
serum  with  positive  and  negative  controls  will  be  de- 
scribed, though  of  course  any  number  of  sera  can  be 
tested  simultaneously,  using  one  set  of  controls. 

Six  small  test-tubes  are  arranged  in  two  rows, 
three  in  each  row.  Tubes  1  of  the  front  and  back  rows 
are  for  the  serum  to  be  tested ;  Tubes  2  for  the  known 
syphilitic  serum;  and  Tubes  3  for  the  non-syphilitic 
serum.  The  back  row  tubes  serve  as  controls  for  those 
of  the  front  row. 

Into  Tube  1  of  the  front  row  are  placed:  0.1  c.c. 
of  patient's  serum;  one  unit  of  complement  (usually 
0.5  c.c.  of  a  1-10  dilution  previously  determined  by 
titration)  ;  one  dose  of  antigen  (usually  0.2  c.c.  of  a 
1-7  dilution). 

Into  Tube  1  of  the  hack  row  are  placed:  0.1  c.c.  of 
patient's  serum ;  one  unit  of  complement ;  no  antigen. 

Tubes  2  and  3  are  dealt  with  in  the  same  way,  ex- 
cept that  0.1  c.c.  of  syphilitic  serum  is  used  in  each  of 
Tubes  2,  and  0.1  c.c.  of  non-syphilitic  serum  in  each 
of  Tubes  3,  instead  of  the  unknown  serum.  The  back 
row  tubes  furnish  controls  to  show  that  the  serum 
alone  without  antigen  will  not  fix  complement.  The 
total  volume  in  each  tube  is  now  made  up  to  4  c.c.  by 
the  addition  of  normal  salt  solution.  The  contents  of 
the  tubes  are  thoroughly  mixed  by  inversion.     The 


FIXATION  OF  COMPLEMENT  123 

finger  can  be  used  as  a  stopper  for  the  tube  while  mix- 
ing, provided  that  contamination  from  one  tube  to 
another  is  avoided  by  using  a  different  finger  for  each 
case.  The  tubes  are  then  incubated  for  one  hour  at 
37°  C.  to  assist  in  fixation  of  complement.  At  the  end 
of  this  time,  no  alteration  in  the  appearance  of  the  con- 
tents is  manifest,  though  complement-fixation  will 
have  taken  place  in  the  tubes  containing  syphilitic 
serum  and  antigen.  In  the  negative  cases  there  will  be 
no  complement-fixation.  The  occurrence  or  non-oc- 
currence of  complement-fixation  is  now  determined  by 
the  addition  of  the  hemolytic  system  and  re-incubation 
of  the  tubes.  To  each  tube  is  added  1  c.c.  of  a  1-1000 
dilution  of  anti-sheep  hemolytic  amboceptor  and  1  c.c. 
of  a  5  per  cent,  suspension  of  sheep's  corpuscles.  The 
contents  of  the  tubes  are  again  thoroughly  mixed,  and 
the  tubes  incubated  for  one  and  a  half  to  two  hours. 
At  the  end  of  this  time  a  preliminary  reading  of  the 
results  may  be  made,  but  final  reading  should  be  de- 
ferred until  next  morning  after  the  tubes  have  re- 
mained all  night  in  the  ice-box.^ 

Reading  of  Besults, — In  case  of  a  positive  result, 
in  Tube  1  of  the  front  row  there  should  be  no  hsemol- 
ysis,  because  complement  being  fixed  by  the  action  of 
the  antigen  on  the  syphilitic  serum,  no  complement  re- 

^  The  time  consumed  in  incubating  the  tubes  may  be  materially  lessened 
by  placing  them  in  a  water-bath  heated  to  37°  C.  instead  of  in  the  air  incubator, 
only  one-half  the  time  being  required  for  each  incubation  in  the  water-bath. 


124  APPLIED  IMMUNOLOGY 

mains  to  take  part  in  hsemolysis  of  the  sheep's  cor- 
puscles by  the  amboceptor.  The  corpuscles  will  have 
settled  to  the  bottom  of  the  tube,  leaving  colorless  fluid 
above.  In  case  of  a  negative  result,  complete  haemol- 
ysis will  have  occurred  in  this  tube,  because  there  being 
no  "  reagin  "  in  the  serum  to  fix  complement  with  the 
antigen,  complement  remains  free  to  act  with  the 
hsemolytic  system  during  the  second  incubation. 
Hsemolysis  is  shown  by  a  disappearance  of  the  cloudi- 
ness due  to  the  corpuscles  and  the  fluid  becoming  a 
transparent  red  color. 

In  Tube  2  of  the  jront  row,  containing  known 
syphilitic  serum,  we  should  get  inhibition  of  hsemol- 
ysis for  the  same  reason  that  we  obtained  it  in  case  of  a 
positive  result  in  Tube  1. 

In  Tuhe  3  of  the  front  row,  containing  non-syphi- 
litic serum,  we  should  get  complete  hsemolysis,  for  the 
same  reason  that  we  obtained  it  in  case  of  a  negative 
result  in  Tube  1. 

In  all  tubes  of  the  hack  row,  there  should  be  com- 
plete hsemolysis,  because  these  tubes  contain  no  anti- 
gen, and  therefore  no  complement-fixation  could  have 
taken  place  during  the  first  incubation,  tlie  comple- 
ment remaining  free  to  combine  with  the  haemolytic 
system.  Occasionally,  in  the  serum  to  be  tested,  there 
are  substances  capable  of  fixing  part  or  all  of  the  com- 
plement in  the  absence  of  antigen.    In  these  cases,  the 


CD 

o 

iO 
U 


Tube  1 


Tube 


RESULT 

Partial  Partial 

iaemolysis      Haemolysis 


Complete 
Haemolysis 


or 


J 

^ediurni-        Weakly -f 
Complete  Haemolysis 


Negative 


T.  .L 


-Ti     ^      .. 


RESULT 


Tube  1  Front 

Patient's  serum 

3 

No                Partial           Partial 
Haemolysis      Hremolysis      Haemolysis 

Complete 
Haemolysis 

row 

0.1  cc. 

c 

Amboceptor 

3 
O 

Complement 

g 

1  cc 

cr 

"■              n              11 

fl 

0.5  cc. 

o 

Corpuscles 

.r\          or         \     .r 

II 

1 

Antigen      serum 

a 

Ice 

5' 

1 

1 

<u 

0.2  cc. 

rr 

3" 

.                f J               1) 

u 

■'-' 

■"^ 

' 

5tronfSlY+        Medium+        Weahl,+ 

Negative 

Q) 

y 

rt 

complete   Hremolysis 

3     Tube    1  BacK 

Patient's    serunn 

Ui 

Amboceptor 

Ol 

<u                     row 

01   cc 

Ice. 

P 

1 

10 

Complement 
0.5  c  c. 

Corpuscles 
Ice 

1 

Tube  Z  Front 

Syphilitic     Serum 

5" 

=" 

Ha  Haemolysis 

ro\A/ 

O.lcc 

c 

Amboceptor 

c 

Complement 

^ 

Ice 

-"I 

0.5  cc. 

5' 

:3 

Corpuscles 

§■ 

.V- 

'o 

Antigen 

Ice 

:t 

i 

§ 

0.2  cc. 

? 

:i 

w 

strong  Iv          -r 

o 

w 

Cu 

Complete  Haemolysis 

>    Tube  2  Back 

5\/philitic    5erum 

Amboceptor 

p 

1 

S                     row 

Oj  cc. 
Complement 

0^ 
P 

Ice 
Corpuscles 

1 

0.5  cc. 

Ice 

u 

Tube   3  Front 

Non-Syphilitic     5erum 

5" 

5" 

.Complete   Haemolysis 

roNA/ 

0.1  cc. 

2 

Amboceptor 

P 

Complement 

* 
'+ 

Ice. 

5 

11 

05  cc 

o' 

3 

Corpuscles 

5 

3 

1 

„ 

Antigen 

Ice. 

_ 

H 

o 

=3- 

V 

1- 

0.2  CO. 

~: 

T^ 

c 

r\ 

N«gatl»e 

u 

a 

.ii..plete    Haemolysis 

1    Tube  3  BacK 

Non-Syphilitic     Serum 

Amboceptor 

Ul 

"IS                   fow 

0.1  c  C. 

p 

Ice 

p 

11 

■z. 

Complement 
05  c  c. 

Corpuscles 
Ice 

\ 

FIXATION  OF  COMPLEMENT  125 

mistake  of  giving  a  positive  result  by  absence  of 
hemolysis  in  the  front  row  tube  is  avoided  by  finding 
a  like  result  in  the  control  tube  in  the  back  row.  In 
reading  the  results,  varying  degrees  of  hsemolysis  may 
be  observed  in  tests  of  different  sera,  ranging  from 
complete  haemolysis  in  negative  cases,  to  absence  of 
haemolysis  in  strongly  positive  cases.  Even  slight  de- 
grees of  inhibition  of  haemolysis  are  generally  to  be  re- 
garded as  positive,  the  closeness  of  reading  of  border- 
line cases  being  dependent  on  clinical  facts  and  the 
experience  of  the  worker.  The  stronger  the  serum  in 
syphilitic  "  reagin,"  the  more  complement  will  be 
fixed,  less  being  left  free  for  haemolysis.  In  a  serum 
containing  smaller  amounts  of  the  Wassermann  sub- 
stance, only  part  of  the  complement  will  be  fixed,  leav- 
ing the  remainder  free,  and  we  get  partial  haemolysis 
(see  Plate  II). 

Quantitative  Estimation, — While  the  degree  of 
haemolysis  produced  gives  an  idea  of  the  strength  of 
the  reaction  up  to  absorption  of  one  unit  of  comple- 
ment, yet  it  does  not  make  any  measure  of  positive 
cases  in  which  the  serum  is  capable  of  absorbing  more 
than  one  unit  of  complement.  In  other  words,  absence 
of  haemolysis  as  seen  by  the  usual  method  means  a 
strongly  positive  reaction,  but  indicates  no  difference 
in  the  degree  of  strength  of  strongly  positive  sera; 
so  that  a  serum  may  show  no  signs  of  weakening  under 


126 


APPLIED  IMMUNOLOGY 


the  effects  of  treatment  when  tested  with  only  one  unit 
of  complement,  because  it  was  previously  capable  of 
fixing  two  or  more  units.  When  we  wish  to  determine 
accurately  the  complement   absorption  power  of  a 


Fig.  14. — Showing  arrangement  of  tubes  in  performance  of  Wassermann  reaction 
on  one  unknown  case,  with  positive  and  negative  controls. 

P.S.I.  Tube  containing  patient's  serum  and  1  unit  of  complement.  P.S.2.  Tube 
containing  patient's  serum  and  2  units  of  complement.  S.C.  Serum  control  tubes, 
without  antigen.  +1.  +2.  Tubes  containing  known  syphilitic  serum  and  1  and  2  units 
of  complement  respectively.  —1,-2.  Tubes  containing  known  non-syphilitic  serum 
and  1  and  2  units  of  complement  respectively.  A.C.  Antigen  control,  containing 
a  double  dose  of  antigen. 

serum,  in  order  to  gauge  the  effects  of  treatment  by 
subsequent  tests,  tubes  should  be  prepared  in  doing  the 
test  containing  two,  three,  or  more  units  of  comple- 
ment in  addition  to  the  usual  one  unit    (Fig.  14). 


FIXATION  OF  COMPLEMENT  127 

By  this  means  we  can  readily  see  whether  the  usual 
amount  of  the  serum  used  is  capable  of  fixing  one,  two, 
three,  or  more  units  of  complement.  It  is  rarely  neces- 
sary to  carry  this  quantitative  determination  beyond 
two  units  of  complement,  though  some  sera  will  fix  five 
or  six  units.  By  means  of  a  series  of  tests  at  intervals 
on  a  case  under  treatment,  we  can  observe  that  the 
serum  gradually  loses  its  power  to  fix  complement,  ab- 
sorbing perhaps  two  units  the  first  time,  then  one  unit, 
then  allowing  hsemolysis  of  half  the  corpuscles,  then 
only  a  few  corpuscles  being  left  unhaemolyzed,  and 
finally  complete  haemolysis  showing  that  the  serum  has 
become  negative.  Some  workers  use  signs  to  desig- 
nate the  strength  of  the  Wassermann  reaction,  a  weak 
reaction  being  indicated  by  +  or  1  plus,  meaning  that 
there  was  only  25  to  50  per  cent,  inhibition  of  haemol- 
ysis, between  50  and  75  per  cent,  inhibition  being  indi- 
cated by  +  +  or  2  plus,  between  75  and  100  per  cent, 
inhibition  as  +  +  +  or  3  plus,  and  complete  inhibition 
as  +  +  +  +  or  4  plus.  The  percentage  of  haemolysis 
cannot  be  thus  accurately  guessed  at  by  the  eye,  and 
moreover  this  method  of  notation  does  not  give  the 
strength  of  the  reaction  beyond  specific  absorption  of 
one  hasmolytic  dose  of  complement.  We  prefer  to 
designate  reactions  up  to  one  unit  of  fixation  as  nega- 
tive, weakly  positive,  medium  'positive,  and  strongly 
positive,  giving  the  number  of  units  of  complement  ab- 


128  APPLIED  IMMUNOLOGY 

sorbed  in  reactions  stronger  than  this.  We  believe 
that  this  method  of  designation  conveys  more  informa- 
tion as  a  rule  to  the  clinician  than  the  plus  signs.  We 
are  indebted  to  Dr.  John  L.  Laird  for  the  principles 
we  follow  in  quantitative  determination  (Pa.  Med, 
Jour.,  1911-12,  XV,  97-102).  For  those  who  wish  to 
measure  the  percentage  of  haemolysis  more  exactly,  the 
Duboscq  colorimeter  may  be  used  according  to  a 
method  described  by  one  of  us  (Ivy,  Jou7\  Amer.  Med. 
Assn.,  Aug.  10,  1912,  p.  432).  We  regard  this  accu- 
rate measurement  as  seldom  called  for,  yet  the  method 
may  be  fomid  useful  to  those  who  are  in  the  habit  of 
guessing  the  percentage  of  haemolysis  in  giving  their 
results. 

The  sera  that  have  been  tested  can  be  preserved 
and  used  as  positive  and  negative  controls  in  future 
reactions,  provided  they  are  reinactivated  by  heating 
before  subsequent  tests  are  made. 

The  table  (Plate  II)  illustrates  the  performance 
of  the  test  on  an  unknown  serum,  with  positive  and 
negative  controls. 

Modifications  of  the  Wassermann  Reaction 

Each  worker  is  apt  to  vary  from  the  original 
technic  in  minor  details  to  suit  his  own  convenience  in 
the  performance  of  the  Wassermann  reaction.  These 
slight  modifications  are  in  no  way  to  be  regarded  as 
radical  changes  in  the  reaction.    By  the  original  Was- 


FIXATION  OF  COMPLEMENT  129 

sermann  reaction,  as  we  view  the  matter,  is  meant 
the  use  of  extract  of  syphiHtic  liver  as  antigen,  an  anti- 
sheep  h^emolytic  system,  fresh  guinea-pig  serum  as 
complement,  and  inactivation  of  the  patient's  serum. 
Various  workers  have  sought  to  improve  on  the  origi- 
nal Wassermann  technic  by  the  use  of  other  antigens, 
as  above  mentioned;  by  using  anti-ox,  anti-pig,  anti- 
human,  and  other  ha^molytic  systems;  by  using  fresh 
instead  of  inactivated  patient's  serum,  etc.  All  at- 
tempts to  simplify  the  technic  have  resulted  in  sacri- 
ficing the  delicacy  of  the  test.  The  modification  that 
is  most  commonly  employed  in  this  country  is  that  of 
Noguchi,  who,  realizing  the  value  of  the  laboratory  re- 
action for  syphilis  to  the  medical  profession  and  the 
training  and  experience  necessary  for  the  performance 
of  the  test  as  laid  down  by  Wassermann,  attempted  to 
simplify  the  technic  so  as  to  make  it  accessible  to  the 
general  practitioner  as  a  routine  office  procedure.  He 
proposed  the  use  of  an  anti-human  heemolytic  system ; 
thus  making  unnecessary  the  use  of  sheep's  blood.  He 
prepared  anti-human  ha^molytic  amboceptor  by  inject- 
ing rabbits  with  human  corpuscles.  The  antigen,  am- 
boceptor, and  complement  were  put  up  in  dry  form  by 
saturating  strips  of  absorbent  paper  with  these  re- 
agents. In  the  performance  of  the  test,  it  was  only 
necessary  to  add  the  antigen  and  complement  papers 
to  diluted  patient's  serum,  incubate,  and  then  add  the 

9 


130  APPLIED  IMMUNOLOGY 

amboceptor  paper  and  washed  corpuscles  from  the  pa- 
tient. But  it  was  soon  found  that  the  complement 
paper  rapidly  lost  its  power  and  became  useless,  lead- 
ing Noguchi  to  discard  it  in  favor  of  fresh  guinea-pig 
serum.  The  antigen  and  amboceptor  papers  are  still 
employed  to  some  extent,  but  accurate  titrations  of  the 
reagents  cannot  be  carried  out  when  they  are  used,  and 
misleading  results  are  liable  to  occur.  Reliable 
workers  employing  the  Noguchi  system  now  use  the 
different  reagents  in  their  original  fluid  form,  titrat- 
ing them  accurately,  so  that  the  technic  as  carried  out 
at  present  has  no  advantage  over  the  original  Wasser- 
mann  in  point  of  simplicity.  Those  accustomed  to  the 
Noguchi  modification  with  careful  titration  of  all  the 
reagents,  no  doubt  obtain  as  reliable  results  as  can  be 
obtained  with  the  Wassermann  technic,  and  even 
claim  that  the  results  are  more  delicate.  We  believe, 
however,  that  by  proper  titration  and  employment  of 
a  minimal  amount  of  complement,  there  will  be  little 
difference  in  the  results  obtained  by  the  two  methods. 
As  in  all  laboratory  procedures,  cases  will  be  en- 
countered on  the  border-line  between  positive  and 
negative  which  can  only  be  judged  by  a  consideration 
of  the  clinical  features  of  the  cases,  and  the  experience 
of  the  worker. 

Hecht'Weinherg      3Iodification. — The      Hecht- 
Weinberg  test  modifies  the  Wassermann  reaction  for 


FIXATION  OF  COMPLEMENT  131 

syphilis  by  utilizing  the  natural  complement  and  anti- 
sheep  hiemolysin  present  in  the  patient's  serum  in- 
stead of  adding  guinea-pig  serum  and  prepared 
heemolytic  amboceptor.  Gradwohl  {Jour,  Amer, 
Med.  Assn.,  July  18,  1914)  regards  this  test  as  of 
great  value  as  a  check  upon  the  Wassermann  reaction. 
In  a  series  of  one  thousand  cases  he  found  the  Hecht- 
Weinberg  was  positive  15  per  cent,  more  often  than 
the  Wassermann. 

Clinical  Application  of  the  Wassermann  Reaction 

In  a  great  many  cases,  the  correct  interpretation  of 
the  examination  of  the  patient's  serum  in  the  labora- 
tory can  only  be  given  when  the  clinical  aspect  of  the 
case  is  considered.  The  clinician  therefore  should  be 
familiar  w^ith  the  meaning  of  a  given  result  from  the 
laboratory  so  that  he  can  intelligently  apply  it  to  the 
case  in  question.  For  instance,  many  practitioners  are 
disappointed  upon  the  receipt  of  a  negative  Wasser- 
mann report  in  a  case  clinically  diagnosed  as  syphilis, 
because  the  blood  has  been  collected  from  the  patient 
when  under  the  influence  of  specific  treatment.  A 
familiarity  w4th  the  following  facts  will  be  of  value 
in  interpreting  the  results  of  the  reaction. 

The  question  as  to  whether  the  Wassermann  reac- 
tion is  specific  for  syphilitic  infection  alone  or  whether 
it  is  found  in  other  diseases,  has  been  the  occasion  for 


132  APPLIED  IMMUNOLOGY 

much  study.  Observers  from  time  to  time  have  ob- 
tained positive  results  in  yaws  or  framboesia,  trypano- 
somiasis, some  cases  of  leprosy,  malarial  fever,  scarlet 
fever,  and  occasionally  in  other  diseases.  It  is  not  sur- 
prising that  a  positive  result  should  be  given  in  yaws, 
a  disease  clinically  similar  to  syphilis  in  many  respects, 
and  which  is  due  to  the  Spirochceta  pertenuis,  an  or- 
ganism very  difficult  to  distinguish  from  the  Tre- 
ponema  pallidum.  Positive  results  with  the  Was- 
sermann  reaction  are  found  more  frequently  in  cases 
of  the  tuberous  type  of  leprosy  than  the  anaesthetic 
form.  In  malaria  the  reports  are  conflicting.  Some 
observers  have  obtained  positive  results  in  a  certain 
percentage  of  cases,  which  become  negative  as  soon  as 
the  malaria  parasites  disappear  from  the  blood. 
Others  have  had  uniformly  negative  results.  Similar 
experiences  have  been  reported  in  scarlet  fever.  After 
summing  up  all  the  data  at  our  disposal,  and  judging 
from  our  own  results,  we  must  conclude  that  except  in 
yaws,  trypanosomiasis,  and  leprosy,  a  positive  Was- 
sermann  reaction  means  that  the  patient  is  the  victim 
of  syphilitic  infection.  The  diseases  named  being 
practically  unknown  in  temperate  climates,  should 
cause  no  confusion  in  this  part  of  the  world.  The 
positive  results  obtained  in  other  diseases  are  either 
due  to  the  fact  that  syphilis  cannot  be  absolutely  ex- 
cluded, or  to  faulty  technic. 


FIXATION  OF  COMPLEMENT  133 

On  the  other  hand,  we  may  obtain  a  negative  Was- 
sermann  reaction  in  the  presence  of  sypliilitic  infection 
under  certain  conditions  to  be  mentioned  below  in 
detail. 

Primary  Stage  of  Syphilis, — The  Wassermann  re- 
action as  a  rule  does  not  become  positive  until  at  least 
two  weeks  after  the  appearance  of  the  chancre,  the 
average  time  being  from  three  to  four  weeks,  though 
cases  have  been  known  in  which  it  was  positive  before 
the  chancre  appeared.  In  our  records  the  earliest 
positive  Wassermann  reaction  was  obtained  four 
days  after  the  appearance  of  the  primary  lesion.  Oc- 
casionally it  does  not  become  positive  until  after  the 
secondary  symptoms  have  manifested  themselves. 
The  Wassermann  reaction  has  proved  that  many  in- 
stances of  so-called  "  soft  sores  "  are  in  reality  due  to 
syphilitic  infection,  or  at  least  a  mixed  infection  exists. 
The  earliest  means  of  making  an  absolute  diagnosis  of 
the  primary  lesion  of  syphilis  is  by  finding  the  Tre- 
ponema pallidum  in  the  secretion  from  the  sore  by 
dark  field  illumination  or  by  stained  smears.  This 
method  fails,  however,  in  a  certain  percentage  of  cases, 
especially  when  local  treatment  has  been  applied;  so 
that,  while  a  negative  Wassermann  reaction  in  the  sus- 
pected primary  stage  of  syphilis  does  not  exclude  the 
disease,  a  positive  reaction  will  frequently  serve  to 
establish  the  diagnosis. 


134  APPLIED  IMMUNOLOGY 

Secondary  Stage. — The  reaction  is  positive  in 
practically  all  cases  of  secondary  syphilis,  and  is  there- 
fore of  the  greatest  value  in  making  the  diagnosis  of 
atypical  cases.  In  this  stage,  too,  we  usually  find  the 
strongest  reactions. 

Tertiary  Stage, — In  tertiary  syphilis  a  positive 
Wassermann  reaction  is  obtained  in  80  to  90  per  cent, 
of  cases.  A  negative  reaction  in  this  stage  therefore 
does  not  have  quite  the  same  significance  as  in  sus- 
pected secondary  syphilis.  Medium  and  weakly  posi- 
tive reactions  are  more  frequent  here  than  in  the 
secondary  stage. 

Latent  Syphilis, — By  latent  stages  are  meant 
periods  in  which  all  symptoms  disappear  for  a  time, 
either  spontaneously  or  as  a  result  of  treatment,  but 
in  which,  as  shown  by  the  Wassermann  test,  the  spe- 
cific reaction  between  the  spirocheetse  and  the  tissues 
is  still  going  on,  indicating  therefore  that  the  disease 
is  not  eradicated.  It  has  been  found  that  persons  in 
this  stage,  although  exhibiting  no  symptoms,  yet  are 
capable  of  infecting  others,  and  themselves  are  liable 
to  develop  later  manifestations  of  the  disease.  It  is 
estimated  that  about  50  per  cent,  of  latent  syphilitics 
give  a  positive  Wassermann  reaction.  It  is  difficult  to 
estimate  this  percentage,  because  we  cannot  always 
say  whether  a  given  case  having  a  negative  Wasser- 
mann reaction  is  cured  or  merely  in  the  latent  stage. 


FIXATION  OF  COMPLEMENT  135 

Craig  and  Xichols  {Jour,  Amer,  3Ied,  Assn., 
1911,  Ivii,  p.  474)  have  found  that  the  recent  ingestion 
of  varying  quantities  of  alcohol  has  a  marked  effect  on 
the  Wassermann  reaction.  These  authors  state  that 
from  180  to  240  c.c.  of  whiskey,  90  c.c.  of  95  per  cent, 
alcohol,  and  700  c.c.  of  Munich  beer  are  capable  of 
causing  a  positive  reaction  to  become  negative  for 
periods  ranging  from  24  to  72  hours.  They  therefore 
recommend,  in  any  case  where  the  ingestion  of  large 
quantities  of  alcohol  in  the  preceding  24  hours  is  sus- 
pected, that  the  removal  of  blood  from  the  patient  for 
performance  of  the  test  be  postponed  for  3  or  4  days. 
The  authors  have  recently  observed  a  case  of  second- 
ary syphilis  in  a  chronic  alcoholic  patient,  whose  Was- 
sermann did  not  become  positive  until  the  seventh 
week  of  the  disease.  Previously,  in  an  extensive  ex- 
perience with  hundreds  of  cases  of  untreated  syphilis, 
the  Wassermann  reaction  always  resulted  positively 
by  the  fifth  week  following  the  appearance  of  the 
chancre. 

It  is  now  pretty  generally  agreed  that  a  positive 
Wassermann  reaction  means  the  presence  of  living 
spiroch^tse  in  the  body,  the  organisms  having  been 
found  in  practically  all  lesions  of  every  stage  of  ac- 
quired and  inherited  syphilis.  The  reaction  differs 
from  true  antigen-antibody  reactions  in  that  the  latter 
are  apt  to  persist  for  some  time  after  the  infecting 


136  APPLIED  IMMUNOLOGY 

agent  has  disappeared  from  the  body,  while  the  Was- 
sermann  "  reagin  "  usually  disappears  very  rapidly. 

In  iriherited  syphilis  the  Wassermann  reaction  is 
positive  in  a  very  high  percentage  of  cases,  at  least  90 
per  cent.  The  reaction  tends  to  show  moreover  that  a 
great  many  congenital  defects  and  maladies  of  young 
infants,  until  recently  regarded  as  due  to  syphilis,  are 
not  caused  by  this  disease.  The  Wassermann  reaction 
has  thrown  considerable  light  on  the  significance  of 
the  laws  of  Colles  and  Prof  eta.  Colles'  law  is  that  the 
mother  of  a  syphilitic  child  may  show  no  signs  of 
syphilis  and  is  immune  to  the  disease.  It  has  been 
found  that  in  a  very  high  percentage  of  such  mothers 
the  blood  gives  a  positive  reaction.  These  cases  proba- 
bly have  syphilis  in  a  modified  form,  and  therefore 
cannot  be  reinfected.  Prof  etas  law  is  that  the  chil- 
dren of  syphilitic  mothers  may  be  apparently  healthy, 
and  yet  not  susceptible  to  syphilitic  infection.  A  high 
percentage  of  these  children  also  give  a  positive  Was- 
sermann reaction,  the  explanation  being  the  same  as  in 
the  case  of  Colles'  law. 

Parasyphilitic  A  Sections, — Under  this  name  are 
grouped  certain  diseases  of  the  nervous  system,  par- 
ticularly paresis  and  tabes  dorsalis.  The  discovery  of 
the  Treponema  pallidum  in  the  lesions  of  paresis  and 
tabes  has  proved  them  to  be  true  manifestations  of 
syphilis  in  the  vast  majority  of  cases.    The  Wasser- 


FIXATION  OF  COMPLEMENT  137 

mann  reaction  may  be  performed  on  cases  of  these  dis- 
eases by  examination  of  either  the  blood-serum  or  the 
cerebrospinal  fluid.  In  using  the  latter  for  the  test 
inactivation  is  not  necessary,  as  the  fluid  contains  no 
complement.  The  technic  is  the  same  as  when  using 
blood-serum,  except  that  0.2  c.c.  of  the  cerebrospinal 
fluid  should  be  employed.  According  to  most  ob- 
servers, in  paresis,  examination  of  the  blood-serum 
shows  the  Wassermann  reaction  to  be  positive  in  prac- 
tically all  cases,  while  the  cerebrospinal  fluid  gives  a 
positive  result  in  about  90  per  cent,  of  cases. 

In  tabes  both  the  blood-serum  and  the  cerebro- 
spinal fluid  give  a  positive  Wassermann  reaction  in  60 
to  70  per  cent,  of  cases. 

In  the  manifestations  of  syphilis  of  the  central 
nervous  system,  which  do  not  differ  materially  from 
syphilitic  lesions  elsewhere,  and  are  grouped  apart 
from  paresis  and  tabes,  the  Wassermann  reaction  with 
the  blood-serum  is  positive  in  practically  all  cases, 
while  it  is  positive  in  only  a  small  percentage  of  cases 
with  the  cerebrospinal  fluid.  In  this  group  are  in- 
cluded gummatous  lesions,  syphilitic  meningitis,  etc. 
From  personal  experience,  we  have  found  in  general 
that  the  cerebrospinal  fluid  is  more  frequently  positive 
or  gives  a  stronger  Wassermann  reaction  than  the 
blood-serum  in  the  majority  of  cases  of  syphilis  in- 
volving the  central  nervous  system. 


138  APPLIED  IMMUNOLOGY 

The  Wassermann  reaction  has  shown  that  a  high 
percentage  of  cases  of  aortic  disease  and  aneurism  are 
syphilitic  in  origin. 

Effects  of  Treatment  on  the  Wassermann  Reac- 
tion,— The  Wassermann  reaction  is  markedly  in- 
fluenced by  treatment.  In  a  syphilitic  undergoing 
treatment  with  mercury  and  particularly  salvarsan, 
the  reaction  may  rapidly  be  reduced  from  strongly 
positive  to  negative.  As  a  guide  or  measure  of  the 
effects  of  treatment,  therefore,  the  Wassermann  reac- 
tion plays  almost  as  important  a  role  as  it  does  in  diag- 
nosis. True  biological  antibodies,  such  as  for  instance 
occur  in  typhoid  fever  as  determined  by  the  agglutina- 
tion reaction,  may  persist  in  the  blood  for  months  and 
even  years  after  the  disease  has  been  eradicated.  On 
the  other  hand,  the  evidence  of  the  Wassermann  reac- 
tion shows  that  the  substances  taking  part  in  it  disap- 
pear from  the  patient's  body  very  shortly  after  the 
destruction  of  the  syphilitic  virus  by  treatment,  or 
when  the  virulence  of  any  remaining  spirochset^  is 
much  diminished. 

In  the  primary  stage,  if  treatment  by  salvarsan  or 
neosalvarsan  be  instituted  as  soon  as  the  diagnosis  is 
made  (by  dark  field  illumination),  the  Wassermann 
reaction  may  never  become  positive  and  secondary 
symptoms  may  never  appear.  With  every  day  that 
treatment  is  delayed,  and  especially  after  the  Wasser- 


FIXATION  OF  COMPLEMENT  139 

mann  reaction  has  become  positive,  a  proportionate 
increase  in  treatment  will  be  required  to  eradicate  the 
disease.  In  the  tertiar}-  stage  it  is  impossible  in  some 
cases  to  render  the  reaction  negative. 

Under  treatment  the  symptoms  usually  disappear 
before  the  Wassermann  reaction  becomes  negative, 
though  occasionally  the  opposite  holds  true;  that  is, 
cases  under  treatment  may  present  a  negative  reaction 
although  symptoms  are  still  manifest.  The  reaction 
may  persist  for  some  time  after  all  symptoms  have 
disappeared,  indicating  that  further  treatment  is 
necessary.  After  being  rendered  negative  by  treat- 
ment, the  reaction  may  after  a  time  again  become 
weakly  positive,  then  gradually  stronger,  and  finally  be 
followed  by  a  reappearance  of  symptoms.  It  is  not 
yet  possible  to  say  when  a  negative  reaction  in  a  case 
that  has  received  treatment  means  that  the  patient  is 
cured  of  syphilis.  If  negative  Wassermanns  have 
been  obtained  at  intervals  of  three  or  four  months  over 
a  period  of  two  years,  during  which  the  patient  has 
had  no  treatment  and  with  no  recurrence  of  symp- 
toms, it  is  probably  justifiable  to  regard  the  case  as 
cured.  But  it  will  require  ten  to  twenty  years'  exper- 
ience with  the  reaction  to  be  positive  that  the  so-called 
parasyphilitic  diseases  or  other  late  manifestations  will 
not  develop  even  after  such  a  series  of  negative  tests. 

JNIuch  closer  readings  of  the  reaction  should  be 


140  APPLIED  IMMUNOLOGY 

made  in  using  it  as  a  guide  to  treatment  than  as  a 
diagnostic  aid.  Border-line  reactions  giving  very- 
slight  inhibition  of  hsemolysis  (5-10  per  cent.)  when 
performed  for  diagnosis  should  be  regarded  as  nega- 
tive. But  such  a  result  in  a  case  that  has  been  known 
to  have  had  syphilis  and  to  have  been  under  treatment 
indicates  that  fm^ther  treatment  is  required. 

Occasionally  in  a  case  of  late  or  treated  syphilis 
giving  a  negative  Wassermann  reaction,  a  positive  re- 
sult may  be  obtained  immediately  after  a  so-called 
provocative  dose  of  salvarsan,  neosalvarsan  or  mer- 
cury. It  is  believed  that  the  treatment  in  such  cases 
is  just  sufficient  to  stimulate  the  spirochsetse  without 
causing  their  death. 

In  view  of  the  fact  that  treatment  hy  mercury  or 
salvarsan  often  produces  a  negative  reaction  in  cases 
where  it  otherwise  would  he  positive,  the  test  should 
not  he  performed  on  a  patient  unless  all  treatment  has 
heen  discontinued  for  at  least  three  weeks.  If  this 
precaution  be  not  observed,  negative  results  will  fre- 
quently be  obtained  in  cases  that  should  be  positive, 
greatly  to  the  surprise  and  chagrin  of  the  practitioner. 

In  employing  the  Wassermann  reaction  in  differ- 
ential diagnosis,  it  must  be  remembered  that  a  positive 
result  does  not  necessarily  mean  that  the  lesion  in 
question  is  syphilitic,  but  that  there  is  syphilitic  infec- 
tion in  the  body  of  the  patient. 


XIII 

FIXATION  OF  COMPLEMENT  (Continued) 

GONOCOCCUS  COMPLEMENT-FIXATION  TEST— SERUM  DIAG- 
NOSIS OF  ECHINOCOCCUS  DISEASE— COMPLEMENT-FIXA- 
TION REACTION  IN  TYPHOID  FEVER— COMPLEMENT- 
FIXATION  REACTION  IN  TUBERCULOSIS— THE  COMPLE- 
MENT-FIXATION REACTION  AS  APPLIED  IN  PROTEIN 
DIFFERENTIATION  (NEISSER-SACHS  REACTION) 

The  Gonococcus  Complement-fixation  Test 
In  recognition  of  the  recent  admirable  work  by 
Schwartz  and  McNeil  on  the  complement-fixation 
test  in  gonococcic  infections,  the  fact  must  not  be  over- 
looked that  Miiller  and  Oppenheim,  in  1906,  were  the 
first  to  apply  this  reaction  to  a  gonorrhoeal  affection 
and  consequently  are  entitled  to  the  distinction  of 
being  termed  the  originators.  The  present  popularity 
of  this  test  has  been  the  outgrowth  of  the  suggestion 
made  by  Schwartz  and  McNeil — namely,  that  of  the 
employment  of  a  polyvalent  antigen.  As  a  result  of 
their  labors,  these  workers  contend,  and  seem  to  have 
proved  conclusively,  through  animal  experimentation : 
(1)  "that  the  different  strains  of  the  gonococcus 
differ  markedly  one  from  another — so  much  so  that 
the  antibodies  produced  in  the  body  by  the  toxin  of  one 
strain  will  in  many  instances  not  bind  the  comple- 
ment in  the  presence  of  an  antigen  prepared  from 
another  strain.  Therefore,  if  only  one  strain  is  used 
in  the  preparation  of  the  antigen,  a  great  many  nega- 

141 


142  APPLIED  IMMUNOLOGY 

tive  results  would  be  obtained  in  positive  cases;  (2) 
an  antigen  prepared  from  many  strains  fixes  the  com- 
plement whenever  one  of  its  component  strains  does 
so,  and  consequently  the  necessity  of  testing  a  serum 
against  a  number  of  antigens  separately  is  avoided. 
It  is  not  to  be  denied  that  there  probably  are  other 
strains  of  gonococci  differing  widely  from  any  pres- 
ent in  the  polyvalent  antigen,  so  that  at  times  a  nega- 
tive result  will  be  obtained  in  a  positive  case." 

While  we  recognize  the  fact  that  a  negative  reac- 
tion may  mean  nothing,  in  fact,  may  be  erroneously 
contradictory,  the  significance,  on  the  other  hand,  of  a 
positive  reaction  has  been  so  great — more  specific,  in 
fact,  than  when  the  lipotropic  antigen,  commonly  em- 
ployed in  the  performance  of  the  Wassermann  reac- 
tion, is  utilized — that  we  have  applied  the  test  in  a  large 
series  of  diverse  cases  with  the  most  gratifying  results. 

Discussion  of  Technic, — Schwartz's  method  and 
the  technic  which  we  practiced  for  a  time  in  compari- 
son with  our  own  gonococcus  complement-fixation 
test  is  as  follows,  so  far  as  the  quantities  of  the  ingre- 
dients participating  in  the  reaction  are  concerned:  (1) 
patient's  serum,  0.02  c.c;  (2)  salt  solution,  sufficient 
to  equalize  the  volume  in  each  tube ;  (3)  antigen  (rou- 
tinely 0.3  c.c.  in  one  tube  and  0.15  c.c.  in  another  tube, 
of  a  commercial  preparation  diluted  1  to  10).  These 
quantities  are  determined  by  preliminary  standardiza- 


FIXATION  OF  COMPLEMENT  143 

tion  with  a  fresh  known  negative  and  a  known  positive 
senun;  the  positive  showing  the  true  antigenic  dose, 
and  the  negative  the  highest  quantity  of  antigen  which 
will  allow  complete  hsemolj^sis ;  in  the  actual  test  this 
large  quantity  of  antigen  is  placed  in  one  tube  and 
one-half  the  quantity  in  a  second  tube;  (4)  comple- 
ment 0.1  c.c.  of  a  dilution  1  to  10;  (5)  amboceptor,  0.1 
c.c.  representing  twice  the  lowest  quantity  that  will 
completely  hsemolyze  0.1  c.c.  of  the  cell  suspension 
with  0.1  c.c.  of  complement  in  one  hour;  (6)  sheep's 
red  blood-cells  (5  per  cent,  suspension),  0.1  c.c.  In- 
cubation is  made  for  one-half  hour  at  37°  C.  in  a 
water-bath  or  for  one  hour  in  dry  heat,  before  and 
after  the  addition  of  the  hsemolytic  system. 

The  technic  on  which  we  have  learned  to  place  the 
greatest  reliance  is  essentially  that  utilized  by  us  in 
the  performance  of  the  Wassermann  reaction,  merely 
substituting  the  gonococcus  specific  antigen  for  the 
syphilitic  lipotropic  antigen,  employing  always  the 
carefully  standardized  single  complement  unit  and 
the  routine  standardization  of  antigen  and  ambocep- 
tor (see  Table  of  Test  Reactions). 

Antigens, — The  necessity  of  a  polyvalent  antigen 
is  indisputable,  presumably  owing  to  the  diversity  of 
the  strains  of  the  gonococcus.  The  only  question  of 
importance  is,  how  may  this  antigen  be  prepared  to 
the  best  advantage?  Schwartz  and  McNeil  in  their 
latest  communication  say  that  the  "  various  strains  of 


144  APPLIED  IMMUNOLOGY 

gonococci  are  grown  on  salt-free  veal-agar,  neutral  in 
reaction  to  phenolphthalein :  24-hour-old  cultures  are 
washed  oiF  the  agar-slants  with  distilled  water  and  the 
resulting  suspension  is  heated  for  two  hours  in  a 
water-bath  at  56°  C.  It  is  then  centrifuged  and 
passed  through  a  Berkefeld  filter.  No  salt  is  added  to 
this  antigen  until  it  is  desired  to  use  it,  when  it  is  made 
up  to  0.9  per  cent,  strength  by  adding  one  part  of  9  per 
cent,  saline  solution  to  nine  parts  of  antigen.  Follow- 
ing Schwartz  and  McNeil's  instructions  to  the  letter, 
we  have  prepared  monovalent,  trivalent  and  hexa- 
valent  antigens  and  have  employed  them  compara- 
tively in  a  large  series  of  cases  with  the  result  that  the 
hexavalent  preparation  gave  the  highest  percentage  of 
positive  results  and  in  every  way  appeared  to  be  the 
most  reliable  of  the  three  antigens.  Even  with  the 
hexavalent  antigen,  prepared  as  described  above,  we 
have  been  forced,  in  comparative  studies,  to  the  con- 
clusion that  it  has  not  been  so  useful  or  reliable  as 
when  prepared  in  the  following  manner :  Forty-eight- 
hour-old  cultures  of  the  same  six  strains  of  gonococci, 
grown  on  blood-agar,  were  washed  off  in  sterile  dis- 
tilled water ;  shaken  for  one  hour ;  and  autolyzed  for 
twenty-four  hours  in  a  thermostat  at  the  temperature 
of  37°  C.  and  heated  in  a  water-bath  at  60°  C.  for 
one-half  hour.  Before  use,  this  antigen  is  diluted  one 
to  ten  by  the  addition  of  0.85  per  cent,  salt  solution. 


FIXATION  OF  COMPLEJVIENT 


145 


Tests  and  con- 
trols for  the 
suspected 
serum 

Tests  for  anti- 
gen    stand- 
ardization 
and  controls 

Tests  for  com- 
p  1  e  m  e  n  t 
standardi- 
sation 

en 

*■           00           to           ^      o      o 

00       ^       05       Cn 

>^     CO     to     H- 

No.  test-tube 

.' 

O          O         Q          o      o      o 
to        to        jj        b     *>     io 

0.05 
0.1 
0.2 
0.3 

c.c. 
0.2? 

0.2? 

0.2? 

0.2? 

Antigen    (dilution 
1:  10) 

bi 

j-i        p        J-        p     H-     h- 
CO         b         CO         b     ►—     ^o 

to       CO       ►f'       bi 

c.c. 
1.3 

1.2 

1.1 

1.0 

NaCl  solution  (0.85 
per  cent.) 

P 

Patient's  serum  (in- 
activated) 

H-                 H*                 h- ' 

o     p     p     p 

.   p 

Known  po.sitive  ser- 
um (inactivated) 

:        :                 P    .°    P 

:      :      :      :  « 

Known  negative  ser- 
um (inactivated) 

p 

0.4? 
0.4? 
0.4? 

0.4 

0.8 

0.4 

0.4? 
0.4? 
04  ? 

o    o    o    o« 

Cr»      rf>.      CO      ioP 

Complement     (dilu- 
tion 1 :  10) 

Incubation  at  37°  C.  for  one  hour                                       |                                         | 

^              i_i                                   n 

Haemolytic  ambocep- 
tor (antisheep) 
(titre=l:2000) 
(dilution  1:1000) 

b 

o        o        b        b     b     b 

o     b     b     b 

b     b     b     oT" 

^     ^     ^     ^« 

Red  blood- corpuscles 
(sheep's  5  per  cent, 
washed  suspension) 

b 

b        b        b        b     b     b 

o     b     b     b 

'o    'o    'o    br* 

Incubation  at  37°  C.  for  one 

and  one-half  hours 

{ 

a> 

1 

Complete  haemolysis.  . 
Complete  haemolysis.  . 
Incomplete  haemolysis 

No  haemolysis  ....... 

(Positive     reaction 
haemolysis=l  unit) 

No  haemolysis 

(Positive    reaction 
haemolysi3=2  units) 

Complete  haemolysis.  . 
(Complete  negative 
reaction) 

Incomplete  haemolysis 
Partial  haemolysis .... 

No  haemolysis 

No  hffimolvsis 

Incomplete  haemolysis 
Incomplete  haemolysis 
Complete  haemolysis . . 
Complete  hsemols'sis. . 

Results 

(immediately  or 

morning  after 

refrigeration) 

ill 

tl 

1 

be  used  in  test  proper. 

To  prove  that  the  antigenic  dose  is  not 
in  itself  anticomplementary. 

To  prove  that  twice  the  antigenic  dose 
is  not  in  itself  anticomplementary. 

To  prove  that  thrice  the  antigenic  dose 
is  not  in  itself  completely  anticom- 
plementary. 

To  determine  quantitatively  the  degree 
of  complement-fixation. 

To  determine  quantitatively  the  degree 
of  complement-fixation. 

Shows  that  there  was  no  immune  body 
present  in  the  patient's  serum  with 
the  aid  of  the  antigen    to    fix    the 

To  determine  quantity  of  antigen  to 

be  used  in  test  proper: 
To  determine  quantity  of  antigen  to 
^  be  used  in  test  proper. 
To  determine  quantity  of  antigen  to 
be  used  in  test  proper. 

To  determine  quantity  of  complement 

to  be  used  in  test  proper. 
To  determine  quantity  of  complement 

to  be  used  in  test  proper. 
To  determine  quantity  of  complement 

to  be  used  in  test  proper. 
To  determine  quantity  of  complement 

to  be  used  in  test  proper. 

O 

f 

s- 

2. 

f 
3 
g 

1 

10 


146  APPLIED  IMMUNOLOGY 

Analysis  of  Cases  Treated. — The  result  of  our 
work  has  been  Httle  more  than  a  corroboration  of  the 
reports  that  have  emanated  from  Schwartz  and 
McXeil  and  those  who  have  confirmed  their  results. 
We  believe,  however,  that,  by  utilizing  the  teclmic 
herein  described,  we  have  added  to  the  accuracy  of  the 
test  as  applied  by  them  and  have  thereby  improved  the 
findings  to  the  credit  of  the  test  and  its  value  in  clinical 
diagnosis.  To  the  increased  positive  results  we  at- 
tribute the  accurate  standardization  of  antigen  on  each 
occasion  and  the  employment  of  a  standardized  single 
complement  unit. 

Reviewing  our  experience  with  the  gonococcus 
complement-fixation  test  in  general,  it  may  be  stated 
that  a  negative  reaction  is  not  decisive  against  the 
presence  of  a  gonorrhoeal  infection,  and  this  is  par- 
ticularly true  during  the  first  six  weeks  of  a  primary 
acute  urethritis  either  anterior  or  posterior  in  the  ab- 
sence of  any  complication,  previous  to  which  time 
we  have  never  obtained  a  positive  reaction;  on  the 
other  hand,  the  supervention,  even  during  the  acute 
stage  of  the  disease,  of  complications  such  as  epididy- 
mitis, arthritis,  prostatitis,  etc.,  is  prone  to  result  in 
the  production  of  a  positive  reaction.  On  the  con- 
trary in  our  experience,  a  positive  reaction  has  been 
pathognomonic  of  a  focus  of  gonococcal  infection  and 
has  assisted  many  times  in  elucidating  obscure  or 


FIXATION  OF  COMPLEMENT  147 

doubtful  lesions.  In  fact,  it  appears  that  the  gono- 
coccus-fixation  test  enjoys  greater  specificity  than 
does  the  Wassermann  reaction,  since  thus  far  we  have 
found  no  alien  infection  or  condition  capable  of  pro- 
ducing a  positive  reaction.  This  much  certainly  can- 
not be  claimed  for  the  Wassermann  reaction.  INIore- 
over,  there  is  no  drug,  as  there  is  in  syphilis,  which  is 
capable  of  causing  the  reaction  to  be  negative  during 
the  existence  of  the  disease.  The  probable  explanation 
for  the  greater  specificity  of  the  gonococcus  comple- 
ment-fixation test  rests  in  the  fact  that  with  gonococcic 
infections  we  employ  a  specific  antigen — the  gono- 
coccus— while  in  the  case  of  syphilis  a  non-specific  or 
lipotropic  antigen  is  employed. 

The  analysis  of  our  cases  further  illustrates 
another  interesting  feature,  namely,  the  persistence  in 
some  cases  for  a  short  time  of  a  positive  reaction,  after 
an  apparent  clinical  cure.  This  has  occurred  so  often 
that  we  no  longer  discharge  a  patient  cured  or  give 
him  a  clean  bill  of  health  so  long  as  he  gives  a  positive 
reaction,  provided  he  has  not  been  the  recipient  of 
immmiotherapy.  Usually  a  persistent  positive  reac- 
tion will  become  negative  in  two  or  three  weeks  follow- 
ing clinical  cure  with  or  without  a  continuation  of 
treatment.  The  only  explanation  is  that  it  requires 
an  indefinite  time  for  the  antibodies,  formed  during 
the  course  of  infection,  to  disappear  from  the  blood. 


148  APPLIED  IMMUNOLOGY 

Torrey,  in  animal  experimentation,  has  found  that  the 
antibodies  in  immunized  rabbits  begin  to  disappear 
after  ten  days,  and  that  the  ehmination  is  practically 
complete  by  the  fiftieth  day  in  all  cases,  disappearing 
much  earlier  in  many  instances.  Thus  a  patient,  evi- 
dencing a  positive  reaction  two  months  after  presumed 
clinical  cure,  should  be  regarded  as  still  harboring  a 
latent  gonorrhceal  focus.  Such  experience,  adopted 
either  as  routine  procedure  in  the  management  of 
treatment,  or  discovered  accidentally  when  gonor- 
rhceal infection  or  its  symptoms  were  denied,  or  dem- 
onstrated by  submitting  suspected  or  positive  syphi- 
litic serum  to  the  gonococcus-fixation  test,  has  been 
encountered  in  a  large  number  of  cases. 

Because  of  the  generally  acknowledged  difficulties, 
at  times,  of  differentiating  the  pelvic  lesions  in  women, 
notably  certain  of  the  inflammatory  from  the  cystic 
and  neoplastic  conditions,  and  also  the  differential 
diagnosis  among  gonorrhoeal,  tuberculous  and  pyo- 
genic infections  themselves,  the  gonorrhoeal-fixation 
test  seems  destined  to  play  a  role.  As  in  the  male,  in 
whom  a  positive  reaction  seems  never  to  occur,  at  least 
so  long  as  the  infection  is  confined  to  the  anterior 
urethra,  so  in  the  female  we  have  been  unable  to  obtain 
a  positive  reaction  unless  the  disease  has  ascended  to 
the  level  of  the  uterus. 

An  interesting,  if  not  important,  feature  connected 


FIXATION  OF  COMPLEMENT  149 

with  this  work  is  the  comparative  importance  and 
value  of  the  serological  and  bacteriological  examina- 
tion of  cases  of  suspected  gonorrhoeal  infection.  It  is, 
to-day,  a  fact  that  the  judiciary  courts  of  our  land  re- 
quire that  the  presence  of  the  gonococcus  be  demon- 
strated, culturally,  in  order  to  establish  its  indisput- 
able and  legal  identity.  Based  on  this  qualification, 
there  are  many  cases  of  gonococcic  infection  impos- 
sible to  determine,  and  we  do  not  hesitate  to  state  that, 
in  our  judgment,  many  such  cultures  are  in  reality  the 
Micrococcus  catarrhalis  and  not  the  gonococcus.  This 
applies  particularly  to  such  isolation  of  the  diplo- 
coccus  of  Neisser  from  chronic  inflammatory  proc- 
esses. JMoreover,  it  must  be  generally  recognized 
that  the  demonstration  of  a  Gram-negative  diplococ- 
cus  in  smear  is  often  insufficient  and  faulty  evidence 
on  which  to  base  a  diagnosis  of  gonococci.  Therefore, 
it  is  most  fortunate  that  in  the  chronic  stage  of  the 
disease  with  complications,  the  complement-fixation 
test  seems  to  be  signally  meritorious,  while  in  the 
acute,  subacute  and  frequently  in  the  chronic  forms  of 
the  diseases  when  the  gonococcus  may  be  demonstrated 
bacteriologically,  the  serological  test  promises  little  or 
nothing. 

Conclusions, — Detailed  and  careful  analysis  of  the 
gonococcus  complement-fixation  test,  performed  with 


150  APPLIED  IMMUNOLOGY 

the  serums  of  the  cases  tabulated  in  our  series/  would 
seem  to  justify  the  following  assertions: 

1.  A  positive  reaction  is  invariably  reliable  and  al- 
ways denotes  the  presence  of  a  focus  of  gonococcic 
infection. 

2.  A  negative  reaction  frequently  occurs  in  the 
presence  of  disease,  especially  in  the  acute  and  sub- 
acute stages  when  the  disease  is  limited  to  the  urethra, 
and  it  is  always  negative  when  the  disease  is  confined 
to  the  anterior  urethra  or  vagina  alone. 

3.  In  no  alien  non-gonorrhoeal  infections  of  sys- 
temic disease  has  a  positive  reaction  been  obtained ;  the 
test,  therefore,  appears  to  be  absolutely  specific. 

4.  A  positive  reaction  has  been  found  to  be  present 
in  21.05  per  cent,  of  patients  clinically  cured.  Such 
patients,  therefore,  should  not  be  discharged  from 
treatment  or  observation  until  a  negative  reaction  has 
been  obtained. 

5.  Not  infrequently,  either  when  suspicious  lesions 
are  presented  or  accidentally,  positive  reactions  will  be 
discovered  in  patients  denying  gonorrhoea. 

6.  In  only  9.09  per  cent,  of  cases  of  acute  and  sub- 
acute antero-posterior  urethritis  has  the  complement- 
fixation  test  resulted  positively.  The  earliest  ap- 
pearance of  a  positive  reaction  in  a  primary  attack  of 

^  Archiv.  Int.  Med.,  January,  1914,  p.  143. 


FIXATION  OF  COMPLEMENT  151 

posterior  urethritis,  without  complication,  occurred  in 
the  sixth  week. 

7.  In  a  number  of  cases  of  chronic  recurrent  ure- 
thritis with  acute  exacerbations,  the  test  was  invaria- 
bly positive ;  many  of  these  patients  undoubtedly  had 
prostatitis. 

8.  The  reaction  resulted  positively  in  one-third  of 
all  cases  of  chronic  posterior  urethritis;  undoubtedly 
many  had  a  mild  or  low-grade  prostatitis. 

9.  In  52.08  per  cent,  of  cases  of  chronic  prostatitis 
a  positive  reaction  was  obtainable. 

10.  Two-thirds  of  all  stricture  cases  demonstrated 
a  positive  test. 

11.  In  epididymitis  a  positive  complement-fixa- 
tion test  was  observed  in  87.5  per  cent,  of  cases.  If, 
from  our  series,  one  case,  probably  tuberculous,  may 
be  eliminated,  and  a  time  duration  of  five  weeks  can  be 
imposed,  the  positive  result  in  this  form  of  disease  has 
been  100  per  cent. 

12.  In  arthritis,  undoubtedly  gonorrhoeal  in  char- 
acter, positive  reactions  were  obtained  in  100  per  cent, 
of  cases. 

13.  In  the  diagnosis  and  differential  diagnosis  of 
pelvic  disease  in  women,  the  gonococcus-fixation  test  is 
destined,  unquestionably,  to  play  an  important  role. 
We  have  been  unable  to  obtain  any  positive  results 
in  uncomplicated  urethritis,  vulvovaginitis  and  Bar- 


152  APPLIED  IMMUNOLOGY 

tholinitis,  and  it  would  appear  that  the  infection  must 
ascend  at  least  to  the  level  of  the  uterus  in  order  to 
produce  a  positive  blood  response. 

14.  Inoculations  of  gonococcus  bacterin,  antigono- 
coccic  serum,  etc.,  may  in  themselves  by  the  produc- 
tion of  immune  bodies  be  causes  of  positive  reactions. 
How  long  these  immunizing  effects  may  endure  is  un- 
known, but  we  have  observed  patients,  treated  by  im- 
munotherapy, who  one  year  later  demonstrated  nega- 
tive complement-fixation  reactions. 

15.  Although  the  bacteriological  demonstration  of 
the  gonococcus  culturally  is  the  only  absolute  method 
for  its  identification  in  chronic  inflammatory  processes, 
the  method  as  a  routine  procedure  is  impractical  and 
susceptible  of  many  failures  and  fallacious  results,  so 
that  the  complement-fixation  test  is  not  only  less  la- 
borious, but  is  productive  of  a  higher  percentage  of 
positive  findings. 

A  series  of  comparative  studies  using  non-specific 
with  the  specific  antigens  in  the  performance  of  the 
gonococcus  complement-fixation  reaction  has  been 
carried  out  (Thomas,  B.  A.,  Ivy,  R.  H.,  and  Bird- 
sail,  J.  C,  Surgery,  Gynecology,  and  Obstetrics, 
1914).  Polyvalent  antigens  were  prepared  from 
various  non-gonorrhoeal  Gram-negative  and  positive 
bacteria,  namely,  the  Micrococcus  catarrhalis,  the  Dip- 
lococcus  meningitidis,  the  Pneumococcus,  the  Strepto- 


FIXATION  OF  COMPLEMENT  153 

COCCUS  pyogenes,  the  Micrococcus  albus  and  aureus, 
the  Colon  bacillus,  and  the  Cory  neb  act  erium,  pseudo- 
diphtheria'. 

From  these  later  studies  we  have  deduced  the  fol- 
lowing facts ;  ( 1 )  In  no  case  have  polyvalent  antigens 
prepared  from  meningococci,  pneumococci,  strepto- 
cocci, staphylococci,  colon  bacilli,  or  corynebacteria 
sufficed  to  fix  complement,  thereby  not  jeopardizing 
the  specificity  of  the  gonococcus  antigen.  ( 2 )  In  ten 
per  cent,  of  sera  examined  a  weakly  positive  result  was 
obtained  with  polyvalent  Micrococcus  catarrhalis  anti- 
gen ;  in  these  cases  the  reaction  was  much  more  marked 
with  the  various  gonococcic  antigens.  Thus  it  may  be 
inferred  that  the  relation  between  the  gonococcus  and 
the  Micrococcus  catarrhalis  is  not  positively  and  abso- 
lutely defined  and  it  is  not  unlikely,  on  the  one  hand, 
that  a  culture  of  the  M,  catarrhalis  is  occasionally  in- 
cluded in  a  supposedly  specific  polyvalent  gonococcus 
antigen,  while,  on  the  other  hand,  it  is  undoubtedly 
true  that  a  mixed  infection  often  due  to  the  M,  catar- 
rhalis exists  in  patients  suffering  from  gonorrhoea  and 
its  complications. 

Serum  Diagnosis  of  Echinococcus  Disease 

Echinococcus  disease  is  rare  in  this  country  in  the 
human  being,  and  therefore  serum  diagnosis  is  seldom 
called  for.    Results  with  the  complement-fixation  test. 


154  APPLIED  IMMUNOLOGY 

however,  have  shown  it  to  be  a  reliable  method  of  diag- 
nosis. The  technic  of  the  reaction  is  the  same  as  that 
for  the  Wassermann  reaction.  The  antigen  used  con- 
sists of  the  fluid  from  hydatid  cysts  of  sheep  affected 
with  the  disease.  Suitable  amounts  of  this  fluid  when 
brought  in  contact  with  the  blood-serum  of  patients 
suffering  from  echinococcus  disease  will  cause  comple- 
ment fixation. 

Complement-fixation  Reaction  in  Typhoid  Fever 
In  reporting  the  results  of  the  complement-fixa- 
tion reaction  in  typhoid  fever,  Garbat  {A?n.  Jr.  Med, 
Sc,  July,  1914)  finds  that  the  serum  of  practically  all 
typhoid  fever  patients  sooner  or  later  gives  a  positive 
complement-fixation  reaction.  A  highly  polyvalent 
antigen  properly  prepared  is  absolutely  essential  in 
order  to  obtain  a  maximum  of  positive  results.  A 
positive  complement-fixation  test  throws  great  cor- 
roborative diagnostic  weight  on  the  side  of  a  doubtful 
or  positive  Widal  reaction.  Occasionally  the  test  is 
positive  before  the  Widal  or  blood  culture,  but  usually 
not  before  the  end  of  the  second  week.  It  generally 
persists  for  about  six  weeks  after  recovery. 

Complement-fixation  Reaction  in  Tuberculosis 
Emplo^^ing  as  antigen  a  simple  emulsion  of  liv- 
ing tubercle  bacilli,  ]McIntosh,  Fildes,  and  RadclifFe 
{Lancet,  Aug.  22,  1914)   obtained  positive  comple- 


FIXATION  OF  COMPLEMENT  155 

ment-fixation  in  70  per  cent,  of  85  pathologically  cer- 
tain cases  and  in  66  per  cent,  of  clinically  certain  cases 
of  tuberculosis.  In  87  controls,  taken  from  normal 
and  disease  conditions,  without  any  selection,  all  were 
negative  but  three,  these  being  two  cases  of  leprosy 
and  one  of  Addison's  disease.  From  these  results  the 
reaction  may  be  regarded  as  highly  specific. 

The  Complement-fixation  Reaction  as  Applied  to 

Protein  Differentiation  (  Neisser-Sachs 

Reaction) 

This  method  may  be  applied  to  supplement  the  re- 
sults of  the  precipitation  reaction,  and  may  serve  to 
differentiate  proteins  where  the  precipitation  reac- 
tion fails.  It  is  of  practical  importance  in  the  medico- 
legal identification  of  human  blood.  An  anti-sheep 
hgemolytic  system  is  prepared  in  the  same  manner  as 
for  the  Wassermann  reaction.  For  the  identification 
of  human  blood,  an  anti-human  serum  is  made  by  in- 
jecting a  rabbit  with  human  blood-serum.  The  anti- 
serum should  be  prepared  of  such  a  strength  that  0.03 
or  0.04  c.c.  will  give  complement  fixation  with  0.00001 
c.c.  of  human  serum.  The  antiserum  having  been 
previously  prepared,  an  extract  of  the  suspected  blood 
is  made  in  approximately  1-1000  dilution  in  the 
same  manner  as  for  the  precipitation  reaction  (see 
Chapter  X). 

The  test  is  carried  out  according  to  the  following 


156 


APPLIED  IMMUNOLOGY 


table,  by  mixing  different  quantities  of  the  extract  of 
the  suspected  blood  with  0.1  c.c.  of  suspected  blood 
and  0.03  c.c.  of  the  antiserum. 


Extract  of  blood 

0.1 

c.c. 

0.05 

c.c. 

0.02 

c.c. 

0.01 

c.c. 

0.005 

c.c. 

0.002 

c.c. 

0.001 

c.c. 

0.0001  c.c. 


Complement 

Antiserum 

0.1  c.c. 

0.03  c.c. 

0.1  c.c. 

0.03  c.c. 

0.1  c.c. 

0.03  c.c. 

.  0.1  c.c. 

0.03  c.c. 

0.1  c.c. 

0.03  c.c. 

0.1  c.c. 

0.03  c.c. 

0.1  c.c. 

0.03  c.c. 

0.1  c.c. 

0.03  c.c. 

The  tubes  containing  the  above  are  placed  in  the 
incubator  at  37°  C.  for  one  hour.  Then  the  doses  of 
hemolytic  amboceptor  and  5  per  cent,  sheep's  cor- 
puscles are  added  and  the  tubes  again  incubated  for 
one  hour.  If  0.01  c.c.  of  the  extract  of  blood  prevents 
haemolysis,  the  test  can  be  regarded  as  positive  for 
human  blood. 

Antisera  for  blood  of  various  animals  can  be  pre- 
pared in  the  same  way  and  the  extract  of  unknown 
blood  tested  with  them. 


XIV 

MISCELLANEOUS  BIOCHEMICAL  REACTIONS 

ABDERHALDEN'S  BIOLOGICAL  TEST  FOR  PREGNANCY— 
SERO-ENZYME  TEST  FOR  SYPHILIS— ABDERHALDEN- 
FAUSER  REACTION  IN  MENTAL  DISEASES— MEIOSTAG- 
MIN   REACTION— EPIPHANIN    REACTION 

Abderhalden*s  Biological  Test  for  Pregnancy 
This  biological  test  belongs  to  a  class  of  newer 
serum  reactions.  It  has  been  found  by  Abderhalden 
of  Halle  that  foreign  proteins  entering  the  blood  pro- 
duce specific  protein-splitting  enzymes.  In  preg- 
nancy, Abderhalden  assumes  that  the  placenta  gives 
off  a  protein  which  causes  the  production  of  a  specific 
enzyme  in  the  blood  which  is  capable  of  splitting  up 
the  placental  protein  with  the  formation  of  peptone 
and  amino-acids.  The  test  consists  therefore  of  incu- 
bating the  serum  of  a  suspected  pregnant  woman 
with  placental  material,  and  then  testing  the  fluid  for 
the  end  products  of  protein  digestion.  Two  methods 
are  employed  in  the  detection  of  these  substances,  first 
the  dialysis  metJiod,  in  which  the  presence  of  dialyz- 
able  peptones  are  tested  for  at  the  end  of  the  incuba- 
tion by  means  of  certain  color  reactions;  and  second 
the  optical  method,  in  which  the  end  products  of  pro- 
tein digestion  are  detected  by  means  of  the  polariscope. 
A  brief   outline   of  the   technic  of   the   dialysis 

157 


158  APPLIED  IMMUNOLOGY 

method  is  as  follows:  Fresh  placenta  is  cut  in  small 
pieces  and  repeatedly  boiled  with  a  little  acetic  acid 
until  all  soluble  protein  is  removed.  This  is  deter- 
mined by  testing  with  the  biuret  reaction.  The  pa- 
tient's serum  is  collected  by  puncturing  a  vein  at  the 
elbow.  Five  or  six  c.c.  of  serum  should  be  obtained, 
and  must  be  absolutely  free  from  haemoglobin.  For 
dialysis,  Schleicher  and  Schull's  diffusion  shells  are 
employed.  These  membranes  must  be  impermeable 
to  the  protein  of  blood-serum  and  permeable  to  pep- 
tone, as  ascertained  by  preliminary  tests.  The  ninhy- 
drin  reaction  is  used  for  reading  the  results.  This 
substance,  the  full  name  of  which  is  triketohydrinden- 
hydrat,  gives  a  blue  color  with  the  end  products  of 
protein  digestion. 

Into  one  of  the  diffusion  shells  are  placed  1 
gramme  of  the  boiled  placental  material  and  1.5  to  2 
c.c.  of  patient's  serum.  The  membrane  is  then  placed 
in  a  small  beaker  containing  20  c.c.  of  water.  The 
fluid  in  and  outside  the  membrane  is  then  covered 
with  toluol  and  the  whole  incubated  at  37°  C.  for  16 
to  24}  hours.  At  the  end  of  this  time,  the  water  in  the 
beaker  is  tested  for  products  of  proteid  digestion  by 
adding  to  it  0.2  c.c.  of  a  1  per  cent,  watery  solution  of 
ninhydrin.  If  the  reaction  is  positive  a  blue  color  re- 
sults. The  reaction  must  be  carefully  controlled  by 
using  at  the  same  time  other  membranes  containing 


BIOCHEMICAL  REACTIONS  159 

serum  alone  and  placenta  alone.  Great  care  must  also 
be  exercised  to  see  that  all  bacteria  are  excluded,  and 
that  the  membranes  are  not  permeable  to  protein,  but 
will  allow  peptone  to  pass  through.  If  the  shells  are 
used  repeatedly,  they  must  be  scrupulously  cleansed 
in  order  to  avoid  contamination  from  previous  tests. 
By  carrying  out  all  proper  precautions,  the  great  ma- 
jority of  observers  have  obtained  results  agreeing  with 
those  of  Abderhalden,  the  test  proving  positive  in  pa- 
tients ranging  from  the  early  weeks  of  intra-  or  extra- 
uterine pregnancy  to  a  few  weeks  post  partum. 
Pearce  and  Williams  {Surgery,  Gynecology ,  and  Ob- 
stetrics, April,  1913,  p.  411)  report  a  series  of  36  cases 
of  pregnancy  giving  positive  results,  controlled  by 
negative  results  in  a  male  and  a  non-pregnant  female. 
Insufficient  work  has  as  yet  been  done  using  normal 
serum  and  serum  of  persons  suffering  with  various 
diseases,  to  place  the  test  on  a  conclusive  basis.  Pearce 
and  Williams  (loc,  cit.)  tested  the  reaction  of  serum  of 
pregnant  women  with  various  organs,  such  as  kidney, 
liver,  and  uterus,  instead  of  placenta,  and  obtained 
positive  results  in  some  cases.  While  the  test  gives 
promise  of  becoming  of  importance  in  the  diagnosis  of 
pregnancy,  yet  further  experiments  to  eliminate  pos- 
sible sources  of  error  must  be  carried  on  before  it  will 
attain  wide  clinical  application.  In  view  of  the  pres- 
ent possible  sources  of  error  in  the  reaction,  and  par- 


160  APPLIED  IMMUNOLOGY 

ticularly  the  difficulty  of  sending  serum  for  a  distance 
that  is  absolutely  haemoglobin- free,  the  advertisements 
of  commercial  laboratories  that  they  are  prepared  to 
carry  it  out  as  a  diagnostic  measure  are  premature. 

The  optical  or  polariscopic  method  of  reading  the 
reaction,  for  which  a  polariscope  is  necessarj^  gives 
practically  the  same  results  as  the  dialysis-ninhydrin 
method,  but  at  present  entails  so  much  expense  that  it 
is  out  of  the  reach  of  the  ordinary  laboratory. 

Abderhalden  and  others  are  at  present  working  on 
similar  protein-splitting  reactions  for  the  diagnosis  of 
cancer  and  other  diseases,  which  promise  much  for  the 
future. 

Pearce  and  Williams  have  worked  out  a  technic 
which  promises  to  do  away  with  many  of  the  serious 
difficulties  of  the  test,  by  which  the  reaction  can  be 
carried  on  in  ordinary  test-tubes  instead  of  the  dif- 
fusion shells.  The  mixture  of  serum  and  placenta  is 
coagulated  by  heat  and  acetic  acid,  and  the  products 
of  protein  digestion,  if  present,  are  then  separated 
from  the  coagulated  serum  proteins  by  filtration. 
Great  caution  must  be  observed  that  coagulation  is 
complete  and  that  the  filtrate  is  rendered  absolutely 
clear  by  a  second  boiling  if  necessary.  This  method 
does  away  in  a  large  measure  with  the  difficulties  due 
to  haemoglobin-stained  sera.  We  quote  the  following 
directions  as  to  the  technic  from  Pearce  and  Williams* 


BIOCHEMICAL  REACTIONS  161 

article:  "Measured  amounts  of  serum  are  placed  in 
several  tubes,  and  to  one  is  added  boiled  placenta,  to 
others  boiled  kidney,  or  heart,  or  whatever  the  control 
may  be,  and  with  one  tube  containing  serum  alone,  and 
others  the  tissues  named  above,  all  are  placed  in  the 
thermostat  at  37 '^  C.  for  24  hours.  At  the  end  of  this 
time  the  contents  of  each  tube  are  poured  into  a  sepa- 
rate beaker,  diluted  with  20  c.c.  of  water,  boiled  with 
the  addition  of  acetic  acid,  and  filtered.  To  10  c.c.  of 
each  filtrate  the  ninhydrin  test  is  applied." 

Jamison  and  Cole  {New  Orleans  Med,  Jour.,  Ixvi, 
3,  p.  188)  using  Pearce  and  Williams'  technic  have 
confirmed  its  reliability  as  compared  with  the  diffusion 
method. 

Sero-enzyme  Test  for  Syphilis 

Baeslack  {Jour,  Amer,  Med.  Assn.,  Mar.  28, 
1914,  p.  1002  and  Aug.  15,  1914,  p.  599)  has  applied 
the  principles  of  Abderhalden's  technic  to  the  diag- 
nosis of  syphilis  in  a  considerable  number  of  cases. 
The  tissues  made  use  of  as  antigen  in  the  reaction  are 
the  pearly  white  gummata  resulting  from  the  inocula- 
tion of  the  testicles  of  rabbits  with  syphilitic  tissue. 
This  tissue  is  prepared  in  the  manner  prescribed  by 
Abderhalden  for  the  placental  tissue  in  the  pregnancy 
test,  and  the  remainder  of  the  technic  is  the  same  as 
that  of  the  Abderhalden  dialysis  method,  using  nin- 
hydrin as  indicator.  Baeslack's  results  correspond 
11 


162  APPLIED  IMMUNOLOGY 

closely  with  those  of  the  Wassermann  reaction  carried 
out  simultaneously.  In  early  cases  of  syphilis  the  sero- 
enzyme  reaction  was  positive  earlier  than  the  Wasser- 
mann. In  cases  of  tabes  and  paresis  in  which  the 
cerebrospinal  fluid  was  tested  the  reaction  was  never 
positive,  showing  that  the  enzyme  is  not  present  in  the 
cerebrospinal  fluid.  The  sero-enzyme  test,  moreover, 
was  positive  in  cases  of  tabes  in  which  the  Wasser- 
mann reaction  of  the  blood-serum  was  negative. 

Abderhauden-Fauser  Reaction  in  Mental  Diseases 

Fauser  {Milnch.  med,  Wochenschr,,  Nov.  18, 
1913,  p.  384)  applied  the  Abderhalden  technic  to  the 
diagnosis  of  certain  mental  diseases,  using  as  antigen 
tissue  from  the  sex  glands.  In  the  case  of  males  he 
employed  testicular  tissue,  and  in  females  ovarian  tis- 
sue. According  to  his  findings,  the  serum  only  of 
patients  with  dementia  pr^ecox  contained  protective 
ferments  against  these  tissues,  which  caused  him  to  re- 
gard the  reaction  as  specific  for  the  diagnosis  of  this 
disease.  Later  workers,  however,  have  found  that 
other  mental  diseases,  such  as  paresis,  manic  depres- 
sive insanity  and  epilepsy,  occasionally  produced  posi- 
tive reactions.  In  a  recent  article,  Simon  {Jour,  A, 
31,  A,j  May  30,  1914,  p.  1701)  concludes  that  a  posi- 
tive reaction,  while  not  specific  for  dementia  precox  as 
held  by  Fauser,  is  the  rule  in  dementia  pr^ecox,  while 


BIOCHEMICAL  REACTIONS  163 

it  is  the  exception  in  purely  functional  psychoses,  and 
that  the  test  promises  to  become  of  great  value  when 
the  technic,  which  at  present  is  open  to  many  errors, 
has  been  perfected. 

Meiostagmin  Reaction 
Ascoli  discovered  that  when  a  bacterial  extract 
and  the  specific  antibody  produced  by  it  in  blood- 
serum  were  brought  together  a  lowering  of  surface 
tension  occurs,  as  shown  by  an  increase  in  the  nmiiber 
of  drops  in  the  fluid.  The  number  of  drops  can  be 
conveniently  measured  by  Traube's  stalagmometer. 
As  an  illustration,  it  may  be  found  that  a  certain  mix- 
ture of  normal  serum  and  extract  of  typhoid  bacilli 
shows  50  drops;  while  a  mixture  of  the  same  quanti- 
ties of  serum  from  a  typhoid  case  and  the  extract  of 
typhoid  bacilli  will  show  52  drops.  This  phenomenon 
has  been  observed  in  the  case  of  several  diseases,  in- 
cluding tuberculosis,  anchylostoma,  and  echinococcus 
disease.  For  all  these  diseases,  however,  there  are 
simpler  clinical  tests  than  the  meiostagmin  reaction. 
This  reaction  promises  to  be  of  some  importance  in 
the  diagnosis  of  malignant  tumors,  particularly  car- 
cinoma. In  this  case  the  serum  of  the  suspected  pa- 
tient is  tested  with  an  extract  made  from  cancer  tis- 
sue. It  has  also  been  found  that  an  extract  made 
from  beef  pancreas  answers  the  purpose  as  well.  An 
extract  of  the  dried  tumor  tissue  or  pancreas  is  made 


164  APPLIED  IMMUNOLOGY 

with  methyl  alcohol  in  the  proportion  of  1  :  4  at  50'' 
C.  for  24  hours.  It  is  then  filtered  while  hot,  and 
again  after  cooling.  The  serum  is  employed  in  1  :  20 
dilution  in  normal  saline  solution. 

In  preliminary  titration  of  the  extract,  decreas- 
ing quantities,  made  up  to  1  c.c.  with  distilled  water, 
are  placed  in  tubes  with  9  c.c.  of  1  :  20  dilution  of  nor- 
mal serum.  A  control  is  also  prepared  with  plain 
water  and  serum  (without  extract).  These  mixtures 
are  incubated  at  37°  C.  for  2  hours,  after  which  the 
number  of  drops  in  each  tube  is  noted  by  means  of  the 
stalagmometer.  For  the  test,  the  dose  of  extract  is 
employed  which  is  in  the  tube  that  contains  3-5  parts 
of  a  drop  more  than  the  control  tube. 

The  following  table  will  illustrate  the  titration  of 
the  extract : 


9  c.c.  normal  serum  (1 
9  c.c.  normal  serum  (1 
9  c.c.  normal  serum  (1 
9  c.c.  normal  serum  (1 
9  c.c.  normal  serum  (1 
9  c.c.  normal  serum  (1 
9  c.c.  normal  serum  (1 
9  c.c.  normal  serum  (1 


20)  4-  1  c.c.  extract 1 

20)  +  1  c.c.  extract 1 

20)  +  1  c.c.  extract 1 

20)  +  1  c.c.  extract 1 

20)  +  1  c.c.  extract 1 

20)  +  1  c.c.  extract 1 

20)  +  1  c.c.  extract 1 


50 
75 
100 
125 
150 
200 
300 


20)  +  1  c.c.  aqua  dest.  (control) 


Number  of  drops 
i^Q  [59  +  3  parts  of  a  drop. 
j-o     59+1  part  of  a  drop. 
o^    59  +  1  part  of  a  drop. 

"d!^    59 

^  03  1  58  +  9  parts  of  a  drop. 

g  £    58  +  8  parts  of  a  drop. 

^  g    58  +  8  parts  of  a  drop. 

.J5  I  58  +  8  parts  of  a  drop. 


In  this  case  the  dose  of  extract  to  be  used  in  the  test 
would  be  1  c.c.  of  the  1  :  100  dilution,  which  shows 
about  3  parts  of  a  drop  more  than  the  control. 

Before  using  the  extract  for  diagnostic  purposes, 
this  dose,  as  determined  by  titration,  should  be  tested 
for  reliability  with  a  number  of  known  carcinomatous 


BIOCHEMICAL  REACTIONS  165 

sera  and  known  negative  cases.  If  it  proves  reliable, 
it  may  then  be  used  for  diagnosis  of  unknown  sera. 
Known  positive  and  negative  controls  should  always 
be  used  in  performing  the  test,  and  titration  of  the 
extract  should  frequently  be  carried  out,  as  its  strength 
is  not  constant.  A  reaction  is  regarded  as  positive 
when  the  number  of  drops  is  more  than  1%  in  excess 
of  the  control. 

For  the  test,  a  mixture  of  the  suspected  serum  and 
the  dose  of  extract  is  made ;  also  a  mixture  of  the  sus- 
pected serum  and  distilled  water.  The  same  thing  is 
done  with  normal  serum.  These  mixtures  are  then 
incubated  for  2  hours  at  37°  C.  In  the  case  of  a  posi- 
tive result  the  mixture  of  the  patient's  serum  and 
tumor  or  pancreas  extract  must  have  an  increase  of 
more  than  1%  drops  over  the  mixture  of  patient's 
serum  and  water,  and  over  the  mixture  of  normal 
serum  and  extract. 

The  following  table  gives  an  example  of  a  positive 
result : 

Number  of  drops 
Normal  serum  (control)  9  c.c.  1  :  20  dil.  +  1  c.c.  extract 

1 :  100  dil 59 

Normal  serum  (control)  9  c.c.  1:20  dil.  +  1  c.c.  aqua  dest.  58  +  4  parts 
Suspected    cancer    serum  9  c.c.  1  :  20  dil.  +  1  c.c.  extract 

1 :  100  dil 61 

Suspected  cancer  serum  9  c.c.  1 :  20  dil.  -\-  1  c.c.  aqua  dest.  58  -|-  7  parts 

Different  investigators  working  with  the  meio- 
stagmin  reaction  give  varying  reports  as  to  its  relia- 


166  APPLIED  IMMUNOLOGY 

bility.  The  general  opinion  appears  to  be  that  while 
it  cannot  be  regarded  as  infallible  in  the  diagnosis  of 
carcinoma,  yet  it  has  a  distinct  clinical  value  when 
taken  along  with  other  tests  and  symptoms. 

Epiphanin  Reaction 
The  epiphanin  reaction  of  Weichardt  is  based  on 
practically  the  same  principle  as  the  meiostagmin  re- 
action, viz.,  an  acceleration  of  diffusion  in  the  fluid 
when  an  antigen  is  brought  in  relation  with  the  specific 
antibody.  SeifFert  has  shown  that  this  phenomenon  is 
manifested  by  a  change  in  reaction  to  phenolphthalein. 
This  test  has  been  applied  especially  to  the  diagnosis 
of  syphilis.  For  this  purpose  0.1  c.c.  of  a  1 :  10  dilu- 
tion of  the  patient's  serum  is  mixed  with  0.1  c.c.  of 
alcoholic  extract  of  syphilitic  fetal  liver,  1  c.c.  of  deci- 
normal  sulphuric  acid  and  1  c.c.  of  an  exactly  equiva- 
lent solution  of  barium  hydroxide  are  slowly  added, 
making  a  neutral  mixture.  On  the  addition  of  a  drop 
of  an  alcoholic  solution  of  phenolphthalein,  the  fluid 
turns  red  if  the  serum  is  syphilitic,  but  does  not  change 
color  if  it  is  non-syphilitic.  It  is  doubtful  if  this  test 
will  become  as  widely  used  as  the  Wassermann  reac- 
tion for  the  diagnosis  of  syphilis. 


XV 

SPECIFIC  BACTERIAL  REACTIONS 

ALLERGIC      PHENOMENA— TUBERCULIN      TESTS— LUETIN, 

GONORRHCEAL    AND    TYPHOID    TESTS— SCHICK'S 

DIPHTHERIA  TOXIN  SKIN  REACTION 

The  interesting  subject  of  hypersusceptibility, 
anaphylaxis  or  aWeTgy  has  been  sufficiently  reviewed 
in  Chapter  TV.  It  will  be  recalled  that  allergy  is 
merely  an  incident  in  the  process  of  immunization  and 
signifies  simply  the  reactive  changes  exhibited  by  an 
individual  after  infection  or  the  injection  of  some 
foreign  sustance  or  antigen.  Allergic  phenomena, 
therefore,  may  be  characterized  as  those  associated 
with  increased  sensitiveness  ( anaphylaxis ) ,  and  those 
with  diminished  susceptibility  (prophylaxis).  It  is 
apparent  that  a  number  of  skin  manifestations  as 
erythematous  and  urticarial  eruptions  of  serum  sick- 
ness, exanthemata  of  eruptive  fevers,  "  disposition  to 
sudden  cuticular  inflammation  "  noted  by  Jenner  in 
his  studies  of  cow-pox  in  1798,  tuberculin  reactions, 
etc.,  are  pure  allergic  phenomena,  since  they  are  the 
exponents  of  localized  foreign  proteins  and  the  toxic 
substances  incident  to  their  destruction  by  the  specific 
antibodies  in  the  blood  of  the  sensitized  body.  Con- 
spicuous among  the  allergic  reactions  to  which  practi- 
cal consideration  at  this  time  will  be  given  are  the 

167 


168  APPLIED  IMMUNOLOGY 

tuberculin  tests,  the  luetin  test  and  the  diagnosis  of 
typhoid  and  gonorrhoea!  infection. 

Tuberculins. — Tuberculin  has  been  prepared  in 
many  ways  and,  although  each  preparation  may  be 
used  diagnostically  as  well  as  therapeutically,  it  is  true 
that  one  furnishes  better  results  for  diagnostic  pur- 
poses, just  as  others  are  superior  from  the  therapeutic 
standpoint.  The  available  products  include  Koch's 
"  old  "  and  "  new  "  tuberculins,  Denys'  "  bouillon  fil- 
trate," Spengler's  "  perlsucht,"  Dixon's  "  bacillary 
extract,"  and  Russian  "  tuberculinum  purum." 

Old  Tuberculin  ("  O.  T.").— The  original  tuber- 
culin made  and  used  by  Koch  has  been  styled  "  old  " 
in  contradistinction  to  his  later  or  "  new  "  tuberculins. 
It  is  prepared  from  the  five  or  six  weeks'  pure  culture 
of  B,  tuberculosis  on  five  per  cent,  glycerin  bouillon. 
The  culture  medium  is  then  evaporated  to  one-tenth 
of  its  volume  and  filtered  through  porcelain.  The 
filtrate  containing  all  the  soluble  secretion  products 
of  tubercle  bacilli  is  then  diluted  with  glycerin,  form- 
ing a  twenty-five  or  fifty  per  cent,  solution.  The 
glycerin  acts  simply  as  a  preservative,  not  as  a  germi- 
cide. Hence  the  stock  solution  may  become  con- 
taminated if  frequently  opened,  and  it  should  be  steril- 
ized before  making  dilutions  by  heating  on  a  water- 
bath  for  one  hour  at  60°  C.  or  by  boiling  in  a  test- 
tube  for  ten  minutes.    It  is  customary  when  making 


SPECIFIC  BACTERIAL  REACTIONS  169 

dilutions  to  use  a  solution  of  approximately  0.25  per 
cent,  phenol  in  normal  saline.  It  is  advisable  to  make 
up  fresh  such  dilutions  about  once  in  two  weeks,  as  the 
phenol  may  cause  some  coagulation  of  the  tuberculin, 
resulting  in  precipitation  and  deterioration.  Dilu- 
tions showing  marked  sedimentation  should  be  dis- 
carded. 

New  Tuberculin  ("  T.  JR."). — Realizing  that  old 
tuberculin  was  merely  a  toxin  of  tubercle  bacilli,  and 
that  the  immunity  produced  by  it  was  to  the  toxin  only 
and  not  to  the  bacterium  itself,  Koch,  in  1897,  seven 
years  after  the  announcement  of  "  O.  T.,"  described 
a  new  tuberculin.  This  consisted  of  the  residue 
(Rilcktand)  of  tubercle  bacilli  and  for  brevity  is 
known  as  "  T.  R."  This  tuberculin  is  prepared  by 
drying  in  vacuo  a  virulent  culture  of  tubercle  bacilli. 
The  dried  substance  is  powdered  in  a  mortar  and 
triturated  with  normal  saline.  After  centrifugation 
the  supernatant  cloudy  fluid  is  discarded.  The  resi- 
due is  again  dried,  ground,  extracted  with  salt  solu- 
tion and  centrif  uged.  This  time  the  clean  supernatant 
fluid  is  pipetted  off  and  retained.  The  process  is  re- 
peated until  the  residue  is  entirely  used  up.  The  clear 
solutions  are  united  and  added  to  glycerin,  forming  a 
twenty  per  cent,  solution.  **  T.  R."  is  standardized 
so  that  one  cubic  centimetre  represents  ten  milli- 


170  APPLIED  IMMUNOLOGY 

grammes  of  the  original  dried  whole  tubercle  bacilli 
or  two  milligrammes  of  the  active  solid  substance. 

New  Tuberculin  ("  B,  E.''), — Appreciating  the 
fact  that  the  injection  of  dead  bacilli,  as  compared 
with  "  T.  R.,"  caused  an  increase  in  the  agglutination 
of  the  blood,  Koch  advocated  a  second  new  tuberculin, 
a  bacillary  emulsion  {Bacillen  Emulsion  or  B,  E,), 
This  is  prepared  by  pulverizing  finely  a  virulent  cul- 
ture of  B,  tuberculosis,  and  suspending  one  part  of  the 
powder  in  one  hundred  parts  each  of  distilled  water 
and  glycerin.  It  is  standardized  so  that  one  cubic 
centimetre  contains  ^ve  milligrammes  of  dried  sub- 
stance in  suspension. 

It  will  be  borne  in  mind  that  the  "  new  tuber- 
culins "  are  suspensions,  not  solutions,  hence  they  must 
be  thoroughly  shaken  before  use.  IMoreover,  therapeu- 
tically, reactions  are  not  so  noticeable  as  after  the  use 
of  "  old  tuberculin  "  and  the  resultant  immunity  is 
greater  and  more  durable,  both  of  which  have  added 
to  their  popularity. 

Denys'  Tuberculin  {'' B,  F"),—ln  1905  Denys 
recommended  the  filtrate  from  bouillon  cultures  of 
tubercle  bacilli.  This  tuberculin  is  commonly  known 
as  Bouillon  Filtrate  or  "  B.  F."  It  is  essentially  the 
same  as  Koch's  old  tuberculin,  differing  only  in  that 
no  heat  is  used  in  its  preparation.  It  contains  all  the 
normal  soluble  products  of  the  B,  tuberculosis. 


SPECIFIC  BACTERUL  REACTIONS  171 

Sjoengler's  Perlsucht  Tuberculin  {''  P.  T.  O/')  .— 
Perlsucht  signifies  *'  pearl  disease  "  in  cattle,  that  is, 
true  bovine  tuberculosis.  Spengler  believed  that  a 
tuberculin  prepared  from  this  strain  of  the  tubercle 
bacillus  would  prove  most  efficacious  in  the  treatment 
of  human  tuberculosis,  and  indeed  such  was  the  case  in 
his  experience.  The  method  of  preparation  of 
Spengler's  tuberculin  is  precisely  identical  with  that 
of  Koch's  old  tuberculin. 

Dixon's  Tuberculin. — This  product  is  a  saline  ex- 
tract of  living  tubercle  bacilli  minus  their  fat.  Six  to 
eight  weeks  old  cultures  from  four  per  cent,  glycerin 
veal  broth  are  removed  and  collected  on  hard  filter 
paper.  Equal  quantities,  by  weight,  of  human  and 
bovine  types  of  bacilli  are  placed  between  sterile  filter 
paper  and  dried  in  a  thermostat  for  twenty-four  to 
forty-eight  hours.  The  dried  bacteria  are  then  treated 
in  an  excess  of  ether  until  all  water  and  glycerin  are 
removed.  Further  extraction  of  the  fat  with  fresh 
ether  is  done  and  the  same  is  removed  from  the  bottom 
of  the  vessel  by  a  Pasteur  pipette.  After  the  bacillary 
mass  has  been  thoroughly  dried  and  freed  from  ether, 
it  is  ground  in  an  agate  mortar  and  suspended  in  nor- 
mal saline  in  the  proportion  of  parts  one  to  five.  The 
suspension  is  then  shaken  for  eight  to  ten  hours,  after 
which  it  is  allowed  to  stand  at  room  temperature  for 
several  days.    Finally  it  is  filtered  until  the  filtrate  is 


172  APPLIED  IMMUNOLOGY 

free  of  bacilli,  as  determined  microscopically,  cultur- 
ally and  by  animal  inoculation.  One  cubic  centimetre 
of  this  extract  represents  one-half  gramme  of  the 
bacillary  mass.  One-half  per  cent,  phenol  is  added  as 
a  preservative. 

Tuherculinum  Purum  {"  T,  P"), — According  to 
"  New  and  Non-official  Remedies,"  this  Russian 
tuberculin  is  "  the  purified  filtered  extract  of  human 
tubercle  bacilli  in  50  per  cent,  glycerin,  prepared  in 
the  same  way  as  in  Koch's  old  tuberculin,  but  subse- 
quently treated  with  alcohol,  ether,  chloroform  and 
xylol  in  order  to  remove  deutero-albumoses."  These 
toxalbumins  and  glycerin-soluble  by-products  of  the 
culture  medium  are  held  responsible  for  a  certain 
amount  of  the  toxic  reaction  noted  in  using  old  tuber- 
culin and  their  elimination  has  resulted  in  this  so- 
called  purified  tuberculin. 

Technic  of  Making  Dilutions 

Many  pharmaceutical  firms  to-day  market  tuber- 
culins in  serial  dilutions  most  convenient  for  immediate 
use.  However,  if  it  be  desirable  or  necessary  to  em- 
ploy stock  preparations,  the  following  technic  may  be 
found  useful : 

Pipettes. —  (a)  One-tenth  c.c.  pipette,  graduated 
in  hundredths,  is  most  economical  of  stock  in  making 
tuberculin  dilutions. 


SPECIFIC  BACTERIAL  REACTIONS  173 

(b)  One  c.c.  pipette,  graduated  in  tenths  for  larger 
dilutions. 

(c)  Ten  c.c.  pipette,  graduated  in  tenths  of  a  c.c. 
for  the  highest  dilutions. 

Pipettes  are  best  sterilized  by  dry  heat  in  specially 
constructed  copper  containers,  or  they  may  be  kept 
immersed  in  a  jar  of  two  per  cent,  phenol  or  alcohol, 
rinsing  in  sterile  diluting  solution  before  use. 

Dilutions. — A  convenient  method  of  making  dilu- 
tions economically  is  as  follows: 

Dilution  No.  1 :   0.1  c.c.  stock  tuberculin,  pipette 

(a)  +  9.9  c.c.  diluting  solution  =  0.001  c.c.  tuberculin. 
Dilution  No.  2:  0.1  c.c.  of  Dilution  No.  1,  pipette 

(b)  (1  subdivision)  +  9.9  c.c.  ==  0.1  c.c.  =  0.00001 
c.c,  tuberculin. 

Dilution  No.  3:  0.1  c.c.  of  Dilution  No.  2,  pipette 
(b)  (1  subdivision)  +  9.9  c.c.  =  0.1  c.c.  =  0.0000001 
c.c.  tuberculin. 

In  doses  of  0.001  c.c.  and  over  it  is  advisable  to 
use  pipette   (b)   for  measuring  the  stock  tuberculin. 

The  Physiological  Action  of  Tuberculin, — Many 
theories  have  been  advanced.  The  most  tenable  ap- 
pears to  be  that  of  Citron,  who  explains  the  presence 
of  antituberculin,  demonstrated  by  Wassermann, 
Bruck  and  Ludke,  in  the  bodies  of  tuberculous  sub- 
jects, on  the  assumption  that  after  an  injection  of 
tuberculin  the  cells  in  the  immediate  vicinity  of  the 


174  APPLIED  IMMUNOLOGY 

tuberculous  focus  unite  with  tuberculin  by  their  re- 
ceptors (Ehrlich),  and  the  cells  thus  attacked  pro- 
duce receptors  in  excess  of  the  demand.  These  over- 
produced receptors  or  antibodies  are  then  set  free  in 
the  serum  to  unite  with  other  portions  of  tuberculin. 
Thus  repeated  tuberculin  inoculations  lead  to  the  for- 
mation of  large  numbers  of  free  agglutinins,  anti- 
tuberculin  and  opsonins  at  the  point  of  local  infection 
as  well  as  many  fixed  receptors  or  antibodies. 

Obviously,  it  will  be  seen  that  the  tuberculin  reac- 
tion is  dependent  upon  the  presence  of  specific  anti- 
bodies. If  the  suspected  individual  is  free  from  tuber- 
culosis and  none  exists,  no  reaction  can  be  produced 
even  by  recourse  to  very  large  doses  of  tuberculin; 
nor  can  a  reaction  be  elicited  in  the  advance  stages  of 
the  disease,  because  all  antibodies  have  been  consumed. 
On  the  other  hand,  in  the  average  case  of  tuberculous 
infection,  a  comparatively  small  dose  of  tuberculin  will 
suffice  to  evoke  a  reaction.  The  typical  tuberculin  re- 
action is  threefold:  general,  focal  and  local.  The 
general  reaction  consists  of  malaise,  headache,  in- 
somnia, bodily  aches,  nausea,  cough,  tachycardia,  and 
particularly  a  rise  in  temperature  of  one  or  more  de- 
grees. These  phenomena  are  probably  due  to  the  fact 
that  tuberculin,  like  any  other  protein,  is  split  up  by 
complement  acting  in  conjunction  with  antibody,  and 
the  split  toxic  products  formed  give  rise  to  the  symp- 


SPECIFIC  BACTERIAL  REACTIONS  175 

toms  as  they  are  eliminated.  The  focal  reaction  con- 
sists of  the  fresh  inflammatory  changes  noted  at  the 
tuberculous  focus,  namely,  congestion,  pain,  tender- 
ness, swelling,  redness,  etc.  The  local  reaction  com- 
prises the  inflammatory  signs  observed  at  the  site  of 
the  injection.  The  focal  and  local  reactions  are  ex- 
plained by  the  interaction  of  combined  tuberculin, 
newly  formed  antibodies  and  complement,  in  attract- 
ing phagocytes,  with  direct  localizing  action,  produc- 
ing thereby  an  inflammatory  reaction.  If  the  local 
reaction  be  severe,  necrotic  tissue  may  be  cast  off. 
Following  the  above  reactions,  there  is  a  tendency  ex- 
hibited by  the  pathological  process  to  heal. 

Tuberculin  as  a  Diagnostic  Agent. — The  employ- 
ment of  tuberculin  in  a  diagnostic  capacity  is  very  ex- 
tensive, and  rightfully  so,  because,  properly  and  com- 
petently utilized,  its  value  at  times  as  an  aid  in 
difl*erential  diagnosis  is  inestimable.  In  the  authors' 
experience  the  positive  or  negative  information  thus 
yielded  has  been  absolutely  dependable.  Its  proper 
use,  however,  entails  the  greatest  caution  and  dis- 
crimination as  to  indications  and  contra-indications  on 
the  part  of  the  patient,  size  and  administration  of 
doses,  and,  not  least  of  all,  the  correct  brand  of  tuber- 
culin. Unanimity  of  opinion  prevails  that  Koch's  old 
tuberculin,  "  O.  T.,"  whatever  may  be  its  method  or 
form  of  application,  is  best  for  diagnostic  purposes. 


176  APPLIED  IMMUNOLOGY 

The  various  methods  utilized  for  the  application  of 
tuberculin  diagnostically  comprise,  (1)  subcutaneous 
injection,  (2)  intradermic  injection,  (3)  cutaneous 
scarification,  (4)  percutaneous  anointment,  and  (5) 
mucous  membrane  instillation. 

Method  of  Subcutaneous  Injection, — First  prac- 
ticed by  Koch,  it  supersedes  in  reliability  any  other 
method  of  tuberculin  application.  Carelessness  may 
render  it  the  most  dangerous  method,  but  properly 
carried  out  it  is  absolutely  harmless.  It  has  been 
variously  modified  as  to  size  and  interval  of  dosage. 
Old  tuberculin  (O.  T.)  is  the  preparation  of  choice. 
Koch  stipulated  that  doses  up  to  250  milligrammes 
could  be  administered  to  perfectly  normal  individuals 
without  reaction,  but  advised  a  limit  of  10  to  25  milli- 
grammes in  practice.  We  feel  that  such  doses  are  en- 
tirely too  large,  and,  with  few  exceptions,  agree  with 
Roth-Schultz  respecting  the  technic  of  inoculations. 
The  subcutaneous  tuberculin  test  is  best  carried  out 
with  the  patient  in  bed  or  at  rest.  His  temperature 
and  pulse  should  be  recorded  every  two  or  three  hours 
for  two  or  three  days,  also  all  clinical  signs  and  symp- 
toms must  be  noted  prior  to  starting  the  first  inocula- 
tion. The  primary  injection  given  is  0.5  milligramme. 
Should  the  slightest  indication  of  a  reaction,  either 
general,  focal,  or  local  (see  page  174),  supervene,  the 
same  sized  dose  is  to  be  repeated  two  or  three  days 


SPECIFIC  BACTERIAL  REACTIONS 


177 


after  its  subsidence.  If  no  sign  of  a  reaction  occurs, 
the  dose  may  be  increased  to  1.25  milligrammes  on  the 
third  day.  Again  if  the  typical  reaction  is  not  pro- 
duced and  merely  suggestive  signs  appear,  the  same 
sized  inoculation  is  repeated,  since  this  may  be  suffi- 
cient to  provoke  a  marked  reaction  after  the  previous 
sensitization.     In  the  absence  of  any  reactive  phe- 


TEMP                                                      MAV 

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Fig.  15. — Edward  G.,  aged  77.  Tuberculous  mastoiditis.  Note  typical  diagnostic 
tuberculin  reaction  after  second  injection  of  tuberculin.  Associated  with  the  febrile 
rise  and  general  reactions  of  malaise  and  headache,  was  a  local  reaction  of  increased 
pain  and  discharge  from  the  mastoid. 

nomena  after  three  or  four  days,  an  injection  of  2.5 
milligrammes  should  be  given.  If  this  fails  to  cause  a 
definite  response,  a  final  maximum  inoculation  of  5 
milligrammes  is  permissible.  In  children,  under  fifteen 
years  of  age,  Baldwin  recommends  doses  of  0.05,  0.2, 
0.5  and  1  milligramme.  A  reaction  to  be  pathogno- 
monic for  any  suspected  lesion  must  comprise,  in 

12 


178 


APPLIED  IMMUNOLOGY 


addition  to  a  rise  in  temperature  of  at  least  one  degree, 
increased  focal  inflammatory  signs,  located  in  the 
lungs,  bones,  joints,  epididymis  or  wherenot  (Figs. 
15  and  16).  Considerable  importance  is  to  be  at- 
tached, also,  to  the  inflammatory  areola  ofttimes  sur- 
rounding the  site  of  injection  (local  reaction).     Ob- 


FiG.  16. — R.  W.  S.  Bilateral  tuberculous  epididymitis.  Observe  the  suggestive 
rise  in  temperature  after  the  first  injection  of  tuberculin,  not  to  be  regarded,  however, 
as  pathognomonic,  owing  to  the  absence  of  any  focal  reaction.  The  reaction  occurring 
on  the  thirteenth  of  the  month  is  typical  generally,  focally  and  locally. 

viously,  a  focal  reaction  outweighs  in  significance  both 
the  general  and  local  reactions,  and  under  such  cir- 
cumstances a  positive  test  may  be  pronounced  even 
though  the  febrile  rise  has  not  amounted  to  a  degree. 
The  sites  commonly  chosen  for  the  injections  are  the 
outer  or  posterior  aspects  of  the  arm,  the  supra-  or 
interscapular  areas,  the  outer  aspects  of  the  thigh  or 


SPECIFIC  BACTERIAL  REACTIONS  179 

leg  and  the  lumbogluteal  regions.  After  cleansing 
the  skin  with  alcohol,  the  needle  is  to  be  introduced  well 
subcutaneously  and  care  taken  not  to  pierce  the 
muscular  sheaths  or  to  enter  a  vein.  The  syringe  best 
adapted  for  making  the  inoculations  is  an  all-glass 
type  graduated  in  minims  or,  better,  fractions  of  a 
cubic  centimetre,  owing  to  its  cleanliness  and  ease  of 
sterilization  (Fig.  17).  Reliable  pharmaceutical 
houses  supply  tuberculin  "  O.  T."  in  serial  dilutions 
ready  for  immediate  use.  If  the  above  technic  be  care- 
fully followed,  and  the  patient  restricted  to  bed  on  the 


''¥7¥^''T'7"4"'wp[HX^ 


Fig.    17. — All-glass  tuberculin  syringe,  graduated  into  hundredths  of  a  cubic  centi- 
metre, particularly  useful  for  both  diagnostic  and  therapeutic  inoculations. 


occurrence  of  a  reaction,  until  its  disappearance,  the 
test  is  devoid  of  danger,  even  when  incipient  and 
latent  pulmonary  tuberculosis  is  present. 

The  subcutaneous  tuberculin  test,  as  stated  above, 
is  indicated  in  all  forms  of  localized  tuberculosis,  when 
the  diagnosis  is  in  doubt.  Among  these  lesions  may  be 
enumerated  tuberculous  ulcerations  of  the  skin,  lym- 
phadenitis, osteitis,  synovitis  and  arthritis,  laryngitis 
and  tracheitis,  phlyctsenular  conjunctivitis,  keratitis, 
iritis,  uveitis,  nephritis,  prostatitis,  seminal  vesiculi- 
tis, epididymitis,  peritonitis  and  pulmonary  tuber- 
culosis. 


180  APPLIED  IMMUNOLOGY 

The  contra-indications  to  the  use  of  the  subcutane- 
ous tuberculin  test  may  be  stated  to  be  general  miliary 
tuberculosis,  acute  phthisis,  advanced  pulmonary 
tuberculosis,  tuberculous  meningitis,  markedly  as- 
thenic individuals  and  convalescents  from  an  acute  ill- 
ness, conditions  in  which  any  tuberculin  application 
may  fail  to  produce  reaction;  in  patients  running  a 
marked  irregular  temperature,  and  possibly  also  in 
children,  the  scarification  or  von  Pirquet  test  takes 
precedence  over  the  method  of  subcutaneous  injection. 

Intradermic  Injection, — Mantoux  has  strongly 
recommended  this  method  for  the  administration  of 
tuberculin,  diagnostically,  and  to  determine,  by  the 
severity  of  the  reaction,  the  sensitiveness  of  the  pa- 
tient prior  to  therapeutic  inoculations.  He  claims 
that  it  is  more  sensitive  than  other  tuberculin  tests. 
Indeed  it  would  appear  too  delicate,  as  almost  all  adults 
show  a  reaction,  although  tuberculosis,  clinically,  is 
absent.  The  test  may  be  serviceable  in  very  young 
children,  but  is  not  destined  to  become  popular.  Man- 
toux employs  0.01  milligranmie  injected  into  the  skin 
of  the  thigh.  The  reaction  is  not  unlike  the  local  in- 
flammatory process  observed  in  the  subcutaneous 
method. 

Scarification  or  von  Pirquet  Cutaneous  Reaction, 
—This  test,  like  the  subcutaneous  method,  depends 
upon  the  antituberculin  in  the  body  fluids  of  the  in- 


SPECIFIC  BACTERIAL  REACTIONS  181 

fected  individual.  The  reaction  is  very  delicate,  thus 
accounting  for  the  high  percentage  of  positive  results. 
The  test  has  its  greatest  applicability  in  children  and 
patients  exhibiting  a  comparatively  high  fluctuating 
temperature.  Old  tuberculin  ("  O.  T."),  again,  is 
the  preferable  preparation  in  the  performance  of  von 
Pirquet's  test.  It  is  conveniently  put  up  in  sealed 
capillary  tubes  and  marketed  by  a  number  of  pharma- 
ceutical firms.  The  technic  is  quite  similar  to  vac- 
cination against  smallpox.  An  area  on  either  the  arm 
or  leg,  usually  the  forearm,  is  cleansed  with  ether,  not 
alcohol  or  soap  and  water,  and  a  drop  of  "  O.  T." 
is  placed  on  the  skin ;  about  two  inches  distant  a  second 
drop  of  glycerin  bouillon,  utilized  as  a  control,  is 
placed.  With  a  lance,  a  needle  or  special  scarifier,  im- 
mersed first  in  the  central  drop,  then  the  drop  of  tuber- 
culin, the  epidermis  is  lightly  scratched,  removing 
only  the  superficial  epithelium  and  avoiding,  if  pos- 
sible, any  evidence  of  blood.  The  drops  should  be  al- 
lowed to  evaporate  to  dryness,  before  applying  a  dress- 
ing of  sterile  gauze,  otherwise  they  may  be  covered 
with  a  vaccine  shield.  At  most  a  very  slight  hyper- 
emia, disappearing  in  twenty-four  to  forty-eight 
hours,  due  to  traumatism,  may  be  noted  at  the  control 
point.  A  typical  positive  reaction  at  the  site  of  the 
tuberculin  drop  is  characterized  by  a  hypersemic  and 
inflammatory  area   from   four  millimetres  to  three 


182  APPLIED  IMMUNOLOGY 

centimetres  in  diameter.  The  intensity  of  the  reaction 
varies  from  a  hyper^emia  with  papule  formation  to 
many  papules  on  an  oedematous  and  inflamed  base, 
to  a  markedly  indurated  zone  exuding  serum  from  the 
scarification  site  ( Plate  III ) .  The  inflamed  area  may 
persist  for  a  week  or  two.  The  reaction  is  not  at- 
tended with  general  fever  or  other  symptoms.  A 
positive  test  simply  indicates  the  presence  of  tuber- 
culosis; it  does  not  specify,  as  does  the  focal  reaction 
in  the  subcutaneous  test,  that  a  suspected  joint,  cor- 
nea, epididymis,  prostate,  etc.,  is  tuberculous.  A  pa- 
tient with  tuberculous  peribronchial  lymphadenitis 
may  exhibit  a  positive  reaction,  while  his  particular 
cause  for  complaint,  a  troublesome  knee,  may  be 
gonorrhoeal. 

Detre  had  evolved  a  modification  of  the  above, 
known  as  a  differential  tuherculin  test  and  therapeutic 
control.  It  has  for  its  objects  the  difl'erentiation  of 
the  human  and  bovine  types  of  infection,  the  measure- 
ment of  the  extent  of  the  disease,  whether  incipient  or 
advanced,  the  selection  of  the  best  variety  of  tuber- 
culin for  immunization  and  a  control  in  therapy 
superior  to  the  opsonic  index.  Three  varieties  of  tuber- 
culin are  employed:  Koch's  "  O.  T.,"  "  B.  F."  prepared 
from  bacilli  of  the  human  type,  and  "  B.  F."  as  in  the 
von  Pirquet  method,  preferably  the  flexor  surface  of 
the  forearm  is  cleansed  with  ether  and  a  pledget  of 


PLATK  III 


^. 


1) 


Von  Pirquet's  cutaneous  tuberculin  test  (positive  reaction). 


SPECIFIC  BACTERIAL  REACTIONS  183 

cotton.  At  distances  of  about  three  inches,  from  above 
downward,  drops  respectively  of  commercial  "  O  T.," 
human  "  B.  F."  and  bovine  "  B.  F."  are  expressed  on 
the  skin  from  capillary  tubes.  In  the  medium  of  each 
drop  the  underlying  skin  is  scarified,  care  being  taken 
to  cleanse  the  scarifier  in  passing  from  one  variety  of 
tuberculin  to  the  other.  The  inoculated  sites  should 
be  inspected  every  twelve  to  twenty-four  hours.  If  no 
reaction  takes  place  in  three  days  the  test  may  be  pro- 
nounced negative.  Positive  reactions  differ  in  no  re- 
spect from  those  described  under  the  von  Pirquet  test. 
Detre  points  out  that  a  greater  reaction  at  the  site  of 
inoculation  with  the  bovine  tuberculin  means  tuber- 
culosis of  that  type  and  indicates  the  use  of  tuber- 
culin of  the  bovine  type  in  therapy ;  also  that  if  greater 
reactions  occur  with  the  Denys  filtrates  than  with 
*'  O.  T.,"  Koch's  old  tuberculin  should  be  employed 
in  immunization  and  vice  versa.  Marked  reactions  are 
usually  observed  in  early  virulent  infections,  although 
not  infrequently  they  occur  in  the  chronic  latent  form 
of  the  disease,  including  surgical  cases.  The  reaction 
to  filtrate  is  prone  to  disappear  more  in  old  chronic 
cases.  Routine  periodic  applications  of  the  test  are 
necessary  if  it  is  to  be  utilized  as  a  control  of  therapy. 
Method  of  Percutaneous  Anointment. — Moro  de- 
scribes the  use  of  an  ointment  compounded  of  equal 
parts  of  tuberculin  "  O.  T."  and  refined  anhydrous 


184  APPLIED  IMMUNOLOGY 

lanolin.  The  test  is  not  so  delicate  or  reliable  as  either 
the  premier  subcutaneous  method  or  even  von  Pir- 
quet's  scarification,  but  is  utilized  by  some  practi- 
tioners, especially  in  febrile  patients,  because  of  its 
ease  of  application  and  absolute  harmlessness.  The 
ointment  is  applied  as  follows :  An  area  of  skin  about 
five  centimetres  in  diameter  on  the  abdomen  or  in  the 
mammary  region  is  cleansed  with  soap  and  water, 
alcohol  and  finally  with  sterile  water  to  remove  all 
traces  of  alcohol.  A  mass  of  the  ointment,  about  the 
size  of  a  pea,  is  thoroughly  rubbed  into  the  prepared 
area  for  one-half  to  two  minutes.  A  gauze  dressing, 
covered  with  oiled  silk  or  wax  paper,  is  serviceable  to 
protect  the  patient's  clothing.  The  reaction,  occur- 
ring usually  within  a  few  to  forty-eight  hours,  rarely 
delayed  for  a  week,  is  characterized  by  a  few  to  a 
hundred  or  more  discrete  papules  from  one  to  five 
millimetres  in  diameter,  in  the  case  of  marked  reac- 
tions surmounting  an  erythematous  base  and  as- 
sociated with  itching,  at  other  times  not.  The  papules 
dry  up  and  desquamate  in  a  few  days  and  at  the  end 
of  two  weeks  merely  a  brownish  pigmentation  re- 
mains visible.  The  reaction  is  not  accompanied  with 
fever,  pain  or  undue  discomfort.  The  more  intense 
reactions  are  observed  in  scrofulous  conditions  and 
bone  tuberculosis ;  weaker  reactions  are  usually  seen  in 
infections  of  the  lungs. 


SPECIFIC  BACTERL\L  REACTIONS  185 

Method  of  Mucous  Membrane  Instillation, — 
Wolff -Eisner  first  reported  on  the  use  of  ten  per  cent. 
"  Alttuberculin  "  as  a  valuable  diagnostic  test  when 
instilled  into  the  conjunctiva.  Almost  synchronously, 
Calmette  pointed  out  the  irritation  due  solely  to  the 
contained  glycerin  in  ordinary  tuberculins,  especially 
'*  O.  T.,"  and  recommended  its  application  as  an  oph- 
thalmic test  only  after  precipitation  and  purification 
with  alcohol.  Even  as  such  the  conjunctival  test  has 
not  been  received  in  all  quarters  with  much  favor, 
owing  to  a  number  of  serious  ophthalmic  complications, 
which  have  arisen  incident  to  the  use  of  tuberculin  in 
the  eye.  Therefore,  it  is  strongly  contra-indicated  in 
conjunctivitis,  ulcers,  blepharitis,  iritis,  keratitis,  tra- 
choma and  all  diseases  of  the  internal  structures  of 
the  eye.  Scrofulous  persons  are  predisposed  to  the 
formation  of  phl^^ctenules  as  a  result  of  the  reaction. 
Tuberculin  for  this  test  is  obtainable,  commercially, 
in  either  solution  or  tablet  form.  Baldwin  advises  an 
initial  instillation  into  the  conjunctival  cul-de-sac  of 
one  minim  of  a  one-half  per  cent,  solution;  in  the 
absence  of  a  reaction  in  forty-eight  hours,  the  same 
quantity  of  a  one  per  cent,  solution  is  instilled  into  the 
other  eye.  The  reaction  usually  develops  in  three  to 
twelve  hours,  but  may  be  delayed  a  day  or  two,  and 
often  endures  for  a  week.  It  is  characterized  by  itch- 
ing, burning,  serofibrinous  exudate,  congestion  and 


186  APPLIED  IMMUNOLOGY 

redness  of  the  caruncle,  sometimes  involving  the  en- 
tire palpebral  conjunctiva  and  lids. 

Vaughan,  Jr.,  states  that  he  has  never  seen  any- 
ill  effects  as  a  result  of  the  application  of  the  test.  His 
technic  consists  in  dissolving  a  tuberculin  tablet  in  five 
minims  of  boiling  hot  water,  producing  a  one  per  cent, 
solution.  One  drop  of  this,  after  cooling,  is  instilled 
into  the  conjunctival  sac;  if  no  reaction  takes  place, 
at  the  end  of  a  week  a  second  instillation  of  one  drop 
of  one-half  per  cent,  solution  is  instilled  into  the  same 
eye.  He  believes  the  zymogen  created  by  the  first  in- 
stillation is  activated  by  the  second,  causing  the  anti- 
tuberculin  ferment  to  attack  the  tuberculin,  thereby 
liberating  the  toxic  cleavage  product  in  large  amount, 
even  though  the  second  dose  is  but  one-half  of  the 
first.  As  soon  as  a  reaction  becomes  distinctive,  the 
conjunctiva  is  copiously  flushed  with  a  solution  of 
boric  acid,  in  order  to  prevent  undue  inflammation. 
This  tuberculin  test  has  been  unsuccessfully  applied  to 
the  nasal  and  vaginal  mucosa. 

JLuetin  Cutaneous  Reaction  in  Syphilis, — Noguchi, 
in  1911,  described  an  allergic  cutaneous  reaction  in 
syphilis.  Following  the  successful  cultivation  of  the 
Treponema  pallidum  {S pirochceta  pallida)  in  ascitic 
fluid  and  ascitic  fluid  agar  containing  pieces  of  pla- 
centa, the  agar  growth  was  ground  to  a  paste  in  a 
mortar,  then  diluted  with  the  fluid  culture,  steriHzed 


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SPECIFIC  BACTERIAL  REACTIONS  187 

at  60°  C.  for  an  hour  in  a  water-bath,  preserved  in 
0.5  per  cent,  phenol  and  standardized  with  the  dark- 
field  microscope,  so  that  forty  to  one  hundred  dead 
treponemata  occupied  the  average  field.  To  this 
preparation  Noguchi  assigned  the  name  luetin,  and 
has  recommended  it  as  a  valuable  diagnostic  aid, 
superseding  even  the  Wassermann  reaction  in  certain 
forms  of  syphilis.  The  technic  of  the  test  consists 
in  sterilizing  a  small  area  of  skin  of  both  arms,  using 
alcoholic  sublimate  solution.  Intradermically,  in  one 
arm,  with  a  syringe  having  a  very  fine  needle,  0.05  of 
a  cubic  centimetre  of  a  control  suspension  is  injected; 
in  the  opposite  arm  a  similar  quantity  of  luetin. 

Normal  or  negative  reactions  consist  of  a  small 
erythematous  and  occasionally  a  papular  formation, 
not  attended  with  pain  or  itching  sensation,  occurring 
in  24  hours,  and  disappearing  usually  in  48  hours  and 
always  in  72  hours,  even  if  papular.  No  indm*ation 
results,  although  yellowish  pigmentation  due  to  ecchy- 
mosis  is  commonly  seen. 

Positive  reactions  assume  three  forms :  ( 1 )  papu- 
lar, (2)  pustular  and  (3)  torpid,  and  are  character- 
ized by  the  formation,  a  day  or  two  after  inoculation, 
of  a  large,  reddish  papule  usually  five  to  ten  millime- 
tres in  diameter  (Plate  IV) .  A  hyperamic  zone  fre- 
quently surrounds  the  papule.  The  inflammatory 
process  increases  and  extends  for  the  next  three  or 


188  APPLIED  IMMUNOLOGY 

four  days,  then  slowly  recedes,  all  signs  disappearing 
usually  in  a  week  or  two.  At  the  height  of  the  reac- 
tion the  papule  commonly  presents  a  bluish-red  colora- 
tion and  may  be  associated  with  vesicles,  even  under- 
going pustulation  and  ulceration  with  secondary  crust 
formation.  In  the  so-called  torpid  form,  the  slight 
reactive  signs  entirely  disappear  in  three  or  four  days 
and  will  suggest  a  negative  result.  However,  after 
ten  or  more  days,  the  injected  site  may  light  up  and 
proceed  to  pustulation  as  above  described.  Systemic 
reactions,  as  malaise,  loss  of  appetite,  diarrhoea  and 
slight  rise  of  temperature  occur  rarely. 

It  is  not  assumed  that  this  test  will  replace  the 
Wassermann  reaction.  Certainly,  the  Wassermann 
reaction  is  infinitely  superior  to  the  luetin  test  in  the 
primary,  secondary  and  acute  tertiary  stages  of  s^^phi- 
lis,  although  in  the  latent  nervous  and  hereditary 
forms  of  the  disease  Xoguchi  adheres  to  the  superior- 
ity of  the  skin  reaction.  He  found  the  test  positive 
in  100  per  cent,  of  manifest  tertiary  lesions,  in  94  per 
cent,  of  latent  tertiar}^  and  in  96  per  cent,  of  heredi- 
tary syphilis.  It  would  seem  that  the  serological  reac- 
tion is  more  readily  influenced  to  become  negative 
under  treatment  than  is  allergic  cutaneous  reaction. 

Cutaneous  Eeaction  in  Gonorrhoea. — In  1908, 
Irons  alluded  to  the  diagnostic  value  attached  to  the 
constitutional  disturbances  provoked  by  therapeutic 


PLATE  V 


X 


\      r 


\ 


tilled  polyvalent  suspension  ol  gonococci  ejection  of  one  cubic  millimetre  of 


SPECIFIC  BACTERIAL  REACTIONS  189 

inoculations  of  dead  gonococci  and  suggested  the  use 
of  gonococcic  bacterin  in  this  capacity.  At  the  time 
of  this  report,  one  of  the  authors  ( Thomas )  had  been 
utihzing  an  alternation  of  subcutaneous  injections  of 
tuberculin,  gonococcic,  streptococcic,  pneumococcic 
and  staphylococcic  bacterins  in  the  differential  diag- 
nosis of  the  etiological  bacterium  in  obscure  articular 
affections,  with  a  view  to  the  determination  of  the 
correct  biological  therapeutic  agent.  Contrary  to 
Irons'  apparent  greater  reliance  on  the  general  and 
focal  reaction,  Thomas  attached  more  significance  to 
the  local  reaction  at  the  injectional  site,  employing 
much  smaller  doses,  100,000,000  instead  of  500,000,- 
000  cocci.  Recently  the  substitution  of  unheated 
bouillon  filtrate  or  a  filtrate  similar  to  old  tuberculin 
for  the  bacterin  has  produced  more  dependable  results. 
Whether  the  filtrate  or  bacterin  is  used  in  small  or 
large  dosage,  subcutaneously  or  intradermically  as  has 
been  done  by  London,  or  by  scarification,  the  reaction 
to  be  looked  for  simulates  precisely  that  observed  in 
tuberculin  tests,  namely,  local  hyperasmic  areas  with  or 
without  papules  or  crusts,  focal  inflammatory  changes 
at  the  affected  site,  malaise,  leucocytosis,  rise  in  tem- 
perature, etc.  The  reaction  appears  in  six  to  twenty- 
four  hours  and  persists  for  two  to  three  days,  usually 
unattended  by  systemic  phenomena,  unless  large  doses 
are  administered.  No  control  is  necessary,  as  relatively 


190  APPLIED  IMMUNOLOGY 

large  inoculations  of  dead  gonococci  do  not  produce 
the  reaction  in  non-gonorrhoeal  subjects.  The  test 
finds  its  greatest  field  of  usefulness  in  the  differential 
diagnosis  of  obscure  synovial,  joint,  periosteal  and 
gjTisecological  affections ;  also  promises  to  be  of  value 
in  the  control  of  candidates  for  matrimony  and  as  a 
guide  to  the  insufficiency  of  treatment. 

Cutaneous  Reaction  of  Typhoid  Immunity, — Gay 
and  Force  very  recently  have  announced  a  decisive 
skin  reaction  in  95  per  cent,  of  persons  who  have  had 
typhoid  fever.  Furthermore,  they  found  that  nine 
out  of  fifteen  individuals  inoculated  from  four  and 
three-fourths  years  to  eight  months  previously,  in  ac- 
cordance with  the  method  of  the  United  States  Army, 
gave  positive  reactions,  and  that  of  twenty-five  per- 
sons inoculated  within  eight  months,  none  showed  a 
negative  response.  They  conclude  that  the  test  may 
serve  as  an  index  of  the  duration  of  typhoid  immunity 
following  protective  inoculation,  thereby  governing 
the  time  for  re-inoculation. 

The  preparation  used  for  the  test  consists  of  ty- 
phoid cultures  grown  for  five  days  in  five  per  cent, 
glycerin-broth.  This  is  evaporated  to  one-tenth  of  its 
original  volume  over  acetone,  simulating  the  prepara- 
tion of  Koch's  old  tuberculin.  The  test  is  carried  out 
precisely  as  is  von  Pirquet's  tuberculin  method,  pro- 
ducing reactions  of  identical  character  (see  page  180)  • 


SPECIFIC  BACTERL\L  REACTIONS  191 

Schick's  Diphtheria  Toxin  Skin  Reaction 

This  test,  which  promises  to  be  of  great  practical 
importance,  has  recently  been  studied  in  this  country 
by  Veeder  (Am.  Jour,  Dis,  Child,,  1914,  viii,  154- 
162).  The  technic  is  similar  to  that  of  the  Noguchi 
luetin  reaction,  and  consists  of  the  intracutaneous  in- 
jection of  an  amount  of  diphtheria  toxin  equal  to  1-50 
of  the  lethal  dose  for  a  250-gramme  guinea-pig.  This  is 
so  diluted  that  the  amount  of  toxin  is  contained  in  0.1 
c.c.  of  fluid.  A  positive  reaction  is  characterized  by  an 
area  of  redness  and  swelling  within  24  or  48  hours, 
and  indicates  the  absence  of  antitoxin  in  the  blood. 
A  negative  result,  on  the  other  hand,  is  evidence  that 
the  individual  is  well  supplied  with  diphtheria  anti- 
toxin. By  actual  test  it  has  been  found  that  in  the  case 
of  a  negative  result  the  individual  tested  possesses  at 
least  0.031  unit  of  antitoxin  per  cubic  centimetre  of 
blood,  which  is  considered  sufficient  to  protect  him 
from  an  ordinary  attack  of  diphtheria. 

The  practical  importance  of  the  test  lies  in  its 
use  as  a  preliminary  indicator  of  the  necessity  of  pro- 
tective injections  of  antitoxin  in  persons  exposed  to 
diphtheria.  It  is  argued  that  those  giving  negative 
reactions  already  possess  in  the  blood  sufficient  anti- 
toxin for  protective  purposes,  and  therefore  require 
no  prophylactic  injection.  Those  giving  positive  reac- 
tions, on  the  other  hand,  possess  insufficient  antitoxin, 


192  APPLIED  IMMUNOLOGY 

and  require  protective  inoculations.  Veeder's  studies 
show  that  in  children  from  birth  to  15  years  of  age, 
which  includes  the  period  of  greatest  liability  to  diph- 
theritic infection,  approximately  50  per  cent,  give 
negative  reactions.  If  the  test  proves  its  reliability, 
this  will  mean  a  saving  of  one-half  of  the  amount  of 
antitoxin  to  be  used  prophylactically,  and  also  a  great 
lessening  in  the  number  of  cases  of  temporary  dis- 
ability that  sometimes  follows  antitoxin  injections. 

Kolmer  and  JNIoshage  {Am.  Jour.  Dis.  Child., 
March,  1915,  p.  189) ,  after  extended  studies  with  this 
reaction,  reach  the  followang  conclusions: 

1.  The  toxin  skin  reaction  is  a  valuable  and  reliable  method  for 
detecting  susceptibility  to  diphtheria. 

2.  Persons  reacting  negatively  to  this  test  usually  contain  at  least 
^Q  unit  of  diphtheria  antitoxin  per  cubic  centimetre  of  serum,  and  this 
amount  of  antitoxin  is  probably  sufficient  to  protect  against  infection. 

3.  Persons  reacting  weakly  or  strongly  positive  usually  contain  less 
than  %o  of  a  unit  of  antitoxin  per  cubic  centimetre  of  serum  or  none  at  all. 
They  may  be  regarded  as  susceptible  to  diphtheria  and  in  the  event  of  ex- 
posure to  infection  should  be  passively  immunized  with  antitoxin  injection. 

4.  About  40  to  50  per  cent,  of  children  ranging  from  1  to  15  years 
of  age  react  positively  to  the  toxin  test;  this  means  that  the  preliminary 
use  of  the  toxin  test  will  eliminate  the  necessity  of  administering  prophy- 
lactic doses  of  antitoxin  to  about  50  per  cent,  of  children. 

5.  The  toxin  reaction  indicates  that  the  immunity  conferred  by  an 
injection  of  antitoxin  begins  to  disappear  after  ten  days  and  has  gen- 
erally passed  away  entirely  after  four  weeks. 

6.  The  increased  susceptibility  of  persons  with  scarlet  fever  to 
diphtheria  is  shown  by  the  toxin  reaction;  even  after  the  injection  of 
antitoxin  about  10  per  cent,  are  susceptible  within  ten  days. 

7.  According  to  the  toxin  reaction  the  immunity  conferred  by  an 
attack  of  diphtheria  is  usually  of  short  duration  or  entirely  absent. 

8.  The  most  practical  application  of  the  toxin  reaction  consists  in 
applying  the  test  as  a  preliminary  measure  to  all  persons  who  have  been 
exposed  to  diphtheria  and  immunizing  only  those  who  react  positively. 


XVI 

TUBERCULIN  THERAPY 

PROPHYLAXIS— THERAPEUTIC  ADMINISTRATION  OF  TU- 
BERCULIN—AVAILABLE PREPARATIONS— MODES  OF 
ADMINISTRATION  AND  DOSAGE— CONTROL  OF  TUBER- 
CULIN TREATMENT— LIMITATIONS  AND  CONTRA-INDI- 
CATIONS— INDICATIONS   AND   RESULTS 

Prophylawis. — Although  the  promises  of  Koch, 
Behring,  Maraghano,  Arloing,  Friedmann  and  others 
have  never  measured  up  to  their  greatest  expectation, 
hope  still  exists  in  the  breasts  of  investigators  that  the 
future  holds  a  specific  for  inoculation  against  tubercu- 
losis. Indeed,  there  appear  to  be  good  reasons,  both 
experimentally  and  clinically,  why  this  should  be- 
come a  realization.  Auto-immunization  in  human 
beings  is  probably  of  common  occurrence.  This  is 
attested  to  by  the  prevalence  of  tuberculosis  in  child- 
hood, and  the  fact  that  only  25  per  cent,  of  patients 
succumb  to  the  disease,  while  in  the  remaining  75  per 
cent,  almost  all  exhibit  evidence  of  healed  tuberculous 
foci.  Thus  it  is  logical,  irrespective  of  the  assertion 
that  tolerance  to  tuberculin  probably  never  means  the 
production  of  true  immunity  in  tuberculosis,  that  we 
should  reinforce  our  defensive  forces,  prophylactically 
as  well  as  therapeutically.  Indeed,  Hamburger,  in 
view  of  the  frequent  contraction  of  tuberculosis  in 
early  childhood,  advises  immunization  in  infancy  and 
asserts  that  the  proper  administration  of  tuberculin  at 

13  193 


194  APPLIED  IMMUNOLOGY 

this  time  constitutes  one  of  its  most  valuable  uses  and 
will  confer  immunity. 

Therapeutic  Administration  of  Tuberculin. — Al- 
though tuberculin  has  failed  to  become  the  much- 
vaunted  panacea  that,  at  first,  it  was  hoped  would  be 
realized,  it,  at  least  in  most  cases,  when  properly  em- 
ployed, favorably  and  decidedly  influences  the  tuber- 
culous process.  This  has  led,  in  recent  years,  to  a 
marked  revival  of  interest  in  its  therapeutic  employ- 
ment. 

It  must  be  clearly  and  definitely  understood  by 
the  clinician,  as  a  fundamental  premise,  that  it  is  not 
claimed  that  tuberculin  is  a  specific  for,  or  per  se  a 
curative  agent  in,  the  treatment  of  tuberculosis.  It  is 
to  be  regarded  simply  as  an  accessory  agent  of  Nature, 
and  when  thus  utiHzed  serves  as  a  most  valuable,  if 
not  indispensable,  adjunct  to  routine  antituberculosis 
measures,  amounting  frequently,  in  localized  and  non- 
febrile  cases,  apparently  to  curative  results.  Failure 
is  due  oftener  to  incompetent,  careless,  or  reckless  ad- 
ministration than  to  tuberculin  itself.  It  must  be  con- 
stantly borne  in  mind  that  tuberculin,  more  so  than 
most  biological  products,  is  a  powerful  agent,  capable 
of  producing  evil,  or  even  disaster,  rather  than  good, 
unless  properly  and  wisely  administered.  In  experi- 
enced hands,  however,  tuberculin  therapy  is  absolutely 
harmless.    Trudeau  states: 


TUBERCULIN  THERAPY  195 

We  have  learned  the  dangers  of  tuberculin  treatment  and  its  evident 
limitations.  We  have,  however,  also  in  late  years  learned  something 
about  the  complex  defensive  resources  of  the  living  organism  which  tend 
to  the  production  of  immunity,  and  how  to  call  them  into  action,  though 
we  are  evidently  as  yet  only  on  the  threshold  of  the  knowledge  of  im- 
munization by  vaccines  in  the  treatment  of  chronic  infections.  Every- 
thing we  know,  however,  points  to  immunization  as  the  goal  toward  which 
our  efforts  should  be  directed.  We  have  much  to  learn  about  tuberculin 
treatment,  but  even  in  the  present  state  of  our  knowledge,  I  am  inclined 
to  think  that  the  production  of  tuberculin  immunity  by  the  mild  clinical 
method  is  capable  of  favorably  influencing  the  course  of  chronic  tuber- 
culosis, of  prolonging  life,  and  in  many  cases  of  aborting  a  commencing 
infection  or  extinguishing  the  smouldering  fires  of  a  chronic  infection. 

Available  Preparations, — It  is  obvious,  from  the 
great  number  of  tuberculins  that  have  been  recom- 
mended during  the  past  twenty-five  years,  that  none 
is  ideal,  particularly  for  therapeutic  purposes.  Some 
have  fallen  into  disuse  because  of  their  greater  toxic 
properties;  others  have  gained  confidence  because  of 
greater  immunizing  power ;  still  others  have  been  sub- 
jected to  purification  processes,  in  the  hope  of  elimi- 
nating toxic  reactions  and  so  on.  Those  preparations 
which  to-day  enjoy  greatest  popularity,  in  the  order 
named,  are  Bacillen  Emulsion  or  "  B.  E,,"  Tuber- 
culin Riickstand  or  "  T.  R.,"  Bouillon  Filtrate  or 
"  B.  F.,"  Old  Tuberculin  or  "  O.  T.,"  Spengler's 
Perlsucht  Tuberculin,  "  P.  T.  O.,"  Dixon's  Bacillary 
Extract,  and  Tuberculinum  Purum,  "  T.  P."  (see 
Chapter  XV) .  The  majority  of  tuberculin  therapists 
favor  Koch's  so-called  new  tuberculins,  "  B.  E."  and 
"  T.  R.,"  believing  that  the  resultant  immunity  is 


196  APPLIED  IMMUNOLOGY 

greater  and  the  toxic  effect  is  less;  some  still  adhere 
to  "  O.  T."  or  its  bovine  preparation  according  to 
Spengler;  a  few  have  faith  in  Dixon's  product  and 
many  have  been  impressed  by  the  virtue  of  "  T.  P.," 
which  Neumann  has  shown  to  be  the  least  toxic,  hence 
to  be  preferred  in  febrile  cases  or  when  the  process  of 
immunization  is  to  be  effected  in  the  shortest  possi- 
ble time.  The  ideal  modus  operandi  would  appear  to 
be  a  combination  or  alternation  of  "  B.  E."  and 
"  B.  F.,"  the  former  consisting  of  the  bacilli  and  their 
extractives  inactivated  by  a  minimal  degree  of  heat, 
the  latter  comprising  the  unheated  filtered  toxins  of 
the  bacilli;  thus  the  patient  would  seem  to  be  im- 
munized against  all  constituents  of  the  bacillus.  A 
number  of  attempts  have  been  made  to  employ  so- 
called  modified  tubercle  bacilli  in  the  treatment  of 
tuberculosis.  The  most  recent  failure  of  this  nature 
was  the  well-advertised  "  Friedmann  Cure  "  or  inocu- 
lation with  non-virulent  turtle  tubercle  bacilli. 

Modes  of  Administration  and  Dosage, — The  usual 
method  of  administering  tuberculin  to  patients  is  by 
subcutaneous  inoculation  (for  technic  see  Chapter 
XV,  p.  178).  In  the  case  of  "  B.  E."  and  "  T.  R.," 
the  common  initial  dose  may  be  0.001  milligramme ;  ^ 
with  Dixon's  tuberculin,  the  beginning  inoculation 

^  A  few  clinicians  utilize  and  recommend  the  superiority  of  infinitesi- 
mal doses  in  tuberculin  therapy,  namely,  an  initial  inoculation  of 
0.0000001  to  0.000000001  milligramme  of  "  B.  E."  or  "  O.  T." 


TUBERCULIN  THERAPY  197 

consists  of  the  extract  from  1.0  milligramme  of  tuber- 
cle bacilli;  with  Tuberculinum  Purum  or  "  T.  P.," 
the  usual  primary  dose  is  0.02  milligramme.  In  the 
absence  of  reactions,  the  size  of  these  doses  is  doubled, 
semi-weekly  and  later  weekly,  until  the  maximum 
tolerant  dose  is  reached.  Dixon  advises  that  the  mini- 
mal dose  be  repeated  five  times  at  intervals  of  five  days, 
before  proceeding  to  the  next  higher  dose,  which  is 
ten  times  the  strength  of  the  first.  This  is  repeated 
five  times  before  giving  dilution  No.  3,  which  is  twice 
the  strength  of  the  former.  Thus  the  patient  is  car- 
ried through  seventeen  more  dilutions,  each  being  an 
increase  of  one-tenth  of  its  predecessor.  In  children 
the  size  of  the  dose  is  regulated  by  the  age  of  the 
patient,  as  in  any  other  therapeutic  remedy.  As  a  rule 
children  tolerate  tuberculin  very  well. 

For  most  tuberculins  the  technic  of  making  dilu- 
tions as  described  in  the  previous  chapter  on  page  172 
will  prove  satisfactory.  Pharmaceutical  firms  now 
prepare  various  tuberculins  in  serial  dilutions,  bearing 
legends  explanatory  of  their  use,  and  rendering  their 
employment  a  matter  of  great  convenience.  If  the 
dilutions  are  not  freshly  prepared,  caution  must  be 
exercised  to  see  that  they  are  not  clouded  or  precipi- 
tated, hence  inert,  due  to  the  action  of  the  preservative 
phenol.  Tuberculins  for  subcutaneous  inoculation  are 
thus  marketed  in  vials  with  hermetically  sealed  rubber 


198  APPLIED  IMMUNOLOGY 

caps,  glass  ampoules,  or  in  tablet  form.  Tuberculin 
tablets,  "  B.  E."  and  *'  T.  R.,"  are  now  obtainable  in 
six  strengths,  namely,  0.0001,  0.001,  0.01,  0.1, 1.0  and 
10  milligrammes.  By  dissolving  a  tablet  in  one  c.c, 
using  a  special  graduated  syringe  (Fig.  17) ,  or  in  a  ten 
c.c.  graduate  containing  ten  c.c.  of  water  or  fractions 
thereof,  injecting  never  more  than  one  c.c,  the  in- 
oculations with  proper  sized  dosage  can  be  readily 
executed.  Tablets  possess  the  advantage  of  being 
more  stable  than  solutions,  although  great  precautions 
as  to  sterility  must  be  observed. 

Contrary  to  the  researches  of  PfeifFer  and  Persch, 
that  pepsin,  trypsin  and  enterokinase  destroy  the  ac- 
tivity of  tuberculin,  a  number  of  investigators  have 
administered  and  recommended  tuberculin  by  mouth, 
stating  that  when  thus  administered  it  is  just  as  effec- 
tive as  by  subcutaneous  injection,  and  carelessly  given 
is  equally  capable  of  harm  and  even  fatal  results. 
Oral  treatment  has  been  carried  out  with  0.00001  to 
0.001  milligramme  of  "  T.  R.  "  in  10  c.c.  of  normal 
horse  serum;  in  the  presence  of  mixed  infection, 
staphylococcic  bacterin  has  been  combined  with  the 
dose,  best  given  on  an  empty  stomach.  Pharmaceu- 
tists have  prepared  triturates  of  "B.  E."  and  "  T.  R.  " 
for  mouth  administration,  the  tablets  arranged  serially 
and  containing  from  0.000001  to  0.01  milligramme 
each. 


TUBERCULIN  THERAPY  199 

Tuberculin  has  also  been  given  per  rectum  either 
in  dosage  of  0.001  milligramme  "  T.  R."  in  normal 
serum  or  in  the  form  of  suppositories  as  recommended 
by  Lissauer.  Suppositories  of  "  O.  T."  are  obtain- 
able, the  doses  ranging  from  one  to  five  hundred  milli- 
grammes. 

Tuberculin  therapy  by  mouth,  and  to  a  less  extent 
by  rectum,  thus  far  has  not  been  and  probably  never 
will  be  very  favorably  received  by  the  profession. 

The  physiological  action  of  tuberculin,  locally, 
focally,  and  generally,  has  been  sufficiently  described 
in  the  foregoing  chapter  (see  page  173). 

Control  of  Tuberculin  Treatment. — Specific  ther- 
apy under  no  circumstances  invites  or  permits  relaxa- 
tion in  the  general  hygienic  and  dietetic  management 
of  the  case. 

Tuberculin  therapy  may  be  controlled  by  (1)  the 
opsonic  index  or  (2)  the  clinical  symptomatology. 

The  opsonic  control  of  Wright  (see  Chapter  XX) 
has  comparatively  few  adherents  in  the  treatment  of 
tuberculosis,  although  Wright  and  his  school  have 
found  it  particularly  well  adapted  to  govern  the  inocu- 
lations in  surgical  tuberculosis.  They  believe  that  so 
long  as  the  opsonic  index  remains  in  the  positive 
phase,  further  and  particularly  increased  dosage  is 
unnecessary  and  inadvisable. 

The  clinical  symptomatology  serves  as  the  popular 


200  APPLIED  IMMUNOLOGY 

method  in  the  control  of  tuberculin  therapy,  not  only 
because  of  the  tedious  technic  of  the  opsonic  index, 
but  also  on  account  of  the  inferior  results  of  the  latter 
method,  at  least  in  the  pulmonary  form  of  the  disease. 
The  practitioner  should  not  be  led  to  believe  that 
tuberculin  treatment,  properly  guided  by  the  clinical 
symptoms,  is  an  easy  matter  requiring  no  special  in- 
struction or  knowledge.  Far  better  that  he  realize 
that  tuberculin  is  a  double-edged  sword,  cutting  suc- 
cess on  the  one  hand  and  strewing  disaster  on  the 
other.  Due  consideration  and  correct  interpretation 
of  trivial  symptoms,  as  malaise,  headache,  slight  fever, 
weakness,  grippy  sensations,  vague  pains,  insomnia, 
anorexia,  nausea,  loss  of  weight ;  slight  focal  reactions, 
as  increased  cough,  expectoration,  rales,  pleuritic  and 
laryngeal  pains,  vesical  irritability,  suppurative  and 
other  inflammatory  signs;  marked  tenderness,  pain, 
redness  and  swelling  at  the  site  of  injection,  are  most 
important  in  therapeutic  inoculations,  and  indicate 
that,  for  the  time  at  least,  the  patient's  tolerance  to 
tuberculin  has  been  reached;  a  further  inoculation  at 
this  time  may  spell  disaster.  High  fever,  prostration, 
marked  focal  inflammatory  signs  and  prolongation  of 
systemic  reactive  phenomena  mean  that  the  patient 
has  received  an  overdose  of  tuberculin  and  that  treat- 
ment must  be  indefinitely  suspended.  Dogmatic  in- 
structions to  increase  progressively  the  dose  every 


TUBERCULIN  THERAPY  201 

three,  five,  or  seven  days,  without  due  regard  to  the 
physiological  effects,  are  criminal.  No  man  should  ever 
employ  tuberculin  who  is  ignorant  of  its  physiologi- 
cal action.  This  action  may  escape  notice,  except  by 
the  skilled  observer.  The  phenomena  which  do  occur 
often  demand  keen  discrimination  as  to  the  size  and  in- 
terval of  subsequent  dosage.  The  patient  should  have 
his  temperature,  pulse  and  respirations  recorded  at 
three-hour  intervals  for  two  or  three  days  before  and 
throughout  the  course  of  treatment.  His  weight  should 
also  be  noted  weekly.  If  febrile  he  must  remain 
quietly  in  bed.  In  general  the  initial  dose  should  be 
quite  small  in  accordance  with  the  directions  given 
above  for  the  particular  tuberculin  concerned.  If  no 
reaction  whatever  be  produced,  the  dose  may  be 
doubled  in  three  or  four  days.  If  there  be  slight  evi- 
dence, general  or  focal,  but  nothing  definite,  the  same 
sized  dose  should  be  repeated.  If,  however,  a  slight 
reaction  occur,  the  next  inoculation  must  not  be  given 
until  all  traces  of  the  reaction  have  disappeared  for 
three  or  four  days,  and  then  not  more  than  half  of 
the  previous  dose  should  be  given.  If,  after  any  in- 
oculation, a  severe  reaction  be  precipitated,  no  further 
inoculation  shall  be  given  for  two  or  three  weeks  after 
the  patient's  condition  has  returned  to  normal,  and  the 
dose  then  must  be  not  more  than  a  fourth  or  a  half  of 


202  APPLIED  IMMUNOLOGY 

the  previous  intolerant  inoculation.  Again  the  dosage 
ascends  the  scale,  and  this  time  will  probably  pass  be- 
yond the  size  of  the  former  intolerant  injection  with- 
out harmful  effect.  Occasionally,  a  patient  will  be  en- 
countered exhibiting  tuberculin  hyper  susceptibility. 
This  does  not  refer  to  the  ordinary  response  to  a  full- 
sized  dose  of  tuberculin,  but  such  a  state  as  is  seen 
when,  after  the  reduction  of  the  dose  to  one-half,  the 
reaction  reappears,  and  after  a  week  or  ten  days  with 
a  further  reduction  to  one-tenth,  an  even  greater  re- 
action occurs.  This  supersensitiveness  to  tuberculin 
can  be  overcome  and  immunization  resumed  by  sus- 
pending all  inoculations  for  three  or  four  weeks,  then 
beginning  far  down  the  scale  of  dosage;  that  is,  one- 
thousandth  of  the  former  inoculation. 

Experience  has  taught  that  most  patients  first 
show  reactions  to  doses  of  tenths  and  hundredths  of  a 
milligramme.  Consequently,  when  doses  of  these  or 
larger  sizes  are  administered,  they  should  be  spaced 
by  a  week  or  ten  days  instead  of  three  or  five  days  as 
is  the  flexible  rule  when  administering  the  early  small 
doses. 

An  all-important  thought  for  the  tuberculin  thera- 
peutist to  bear  constantly  in  mind  is  that  in  the  average 
case  it  will  require  six  to  eight  months  to  reach  large 
immunizing  doses;  if  the  patient  be  hypersensitive  the 


TUBERCULIN  THERAPY  203 

time  must  be  extended  to  a  year  or  more.^  Any  at- 
tempt to  "  push  the  treatment,"  by  shortening  the 
intervals  or  carelessly  increasing  the  size  of  the  doses, 
will  result  in  failure. 

Limitations  and  Contra-indications  of  Tuberculin 
Therapy. — Two  facts  must  be  emphatically  and  in- 
delibly impressed  in  the  mind  of  the  tuberculin  thera- 
peutist. They  are:  First,  that  tuberculin  acts  simply 
as  an  accessory  to  nature,  and,  second,  that  tolerance, 
even  to  large  doses  of  tuberculin,  does  not  necessarily 
confer  immunity  against  tuberculosis.  If  these  points 
are  fully  realized,  tuberculin,  in  capable  hands,  will 
measure  up  to  expectations  and  be  accorded  its  right- 
ful place  in  modern  therapeutics. 

Success  in  tuberculin  therapy  is  directly  propor- 
tionate to  the  degree  of  dosage  attainable  without  pro- 
ducing deleterious  reactions  or  intolerance.  In  the 
average  case  this  is  a  procedure  of  months'  or  perhaps 
years'  duration,  in  the  event  of  the  necessity  of  inter- 
mittent administration.  Obviously,  little  can  be  ex- 
pected from  a  short  course  of  tuberculin  inoculations 
with  a  maximum  tolerant  dose  of  only  a  fraction  of  a 
milligramme. 

After  the  patient  has  been  successfully  carried 

^  Tuberculinum  Purum,  "  T.  P.,"  is  an  exception  to  this  doctrine,  since 
the  patient  may  receive  the  entire  series  of  inoculations  in  the  course  of 
three  or  four  months,  due  to  the  lesser  toxicity  of  this  preparation,  making 
it  possible  to  ascend  the  scale  of  doses  rapidly. 


204  APPLIED  IMMUNOLOGY 

through  a  course  of  treatment  and  has  reached  large 
doses,  these  must  not  be  continued  indefinitely,  lest 
the  patient  become  over-stimulated  and  thrust  into  the 
''  negative  phase,"  hence  no  longer  capable  of  the  pro- 
duction of  specific  antibodies.  It  were  better  to  cease 
inoculations  for  several  months,  when,  if  indications 
arise,  a  new  course  of  treatment  may  be  instituted. 

As  a  general  rule,  tuberculin  therapy  is  contra- 
indicated  in  acute,  diffuse  and  chronic  affections  with 
acute  exacerbations.  It  is  far  more  applicable  to  so- 
called  surgical  or  localized  tuberculosis  than  to  the 
pulmonary  form  of  the  disease.  In  short,  it  may  be 
stated  that  tuberculin  should  not  be  employed  in  phthi- 
sis pulmonalis  except  in  the  afebrile  or  mildly  febrile 
cases.  It  is  definitely  contra-indicated  when  a  remedia- 
ble surgical  condition,  as  caries,  sequestrum,  tubercu- 
lous kidney,  etc.,  exists,  until  after  operative  interven- 
tion. The  usual  acute  or  advanced  case  of  pulmonary 
involvement  should  be  invariably  treated  by  rest  in  con- 
junction with  the  well-known  climatic,  dietetic,  and 
hygienic  antituberculosis  measures.  Subsequently,  if 
there  be  no  contra-indication  and  the  patient  is  not 
progressing  satisfactorily,  tuberculin  should  supple- 
ment the  treatment.  Tuberculin  therapy  is  decidedly 
contra-indicated  in  debilitated  or  wasted  individuals 
or  patients  exhibiting  very  active  lesions  or  extensive 
complications,  characterized  by  considerable  and  in- 


TUBERCULIN  THERAPY  205 

creasing  fever,  although  shght  dyspnoea,  increased 
respirations,  accelerated  pulse-rate  and  even  hsemop- 
tysic  tendencies,  with  or  without  evidences  of  dry  pleu- 
ritis,  are  not  necessarily  contra-indications.  Extreme 
caution  must  be  exercised  in  practicing  tuberculin  in- 
oculations on  ambulatory  patients,  and  particularly 
is  this  so  if  they  are  febrile,  even  though  slightly  and 
only  occasionally.  Menstruation  and  the  supervention 
of  an  acute  infection  as  a  "  cold,"  tonsillitis,  bronchitis, 
etc.,  are  definite  indications  for  the  temporary  suspen- 
sion of  bacterial  inoculations. 

A  matter  of  some  importance  in  tuberculin  therapy 
is  the  prevalence  of  mioced  infection  in  tuberculous 
processes,  either  pulmonary  or  surgical.  The  advent 
of  the  secondary  invading  bacterium  is  characterized 
by  an  increased  and  typical  irregularity  of  tempera- 
ture. Under  such  circumstances  culture  and  reculture 
of  the  suppuration  from  time  to  time  and  the  prepara- 
tion and  administration  of  autogenous  bacterins,  pre- 
ceding or  alternating  with  tuberculin,  will  produce 
results  not  obtainable  with  tuberculin  alone. 

Indications  and  Results  of  Tuberculin  Therapy 
in  Tuhercidosis, — Tuberculin  is  a  powerful  therapeu- 
tic agent  in  all  localized,  subacute  and  chronic  forms 
of  tuberculosis,  not  excluding  the  pulmonary  type, 
provided  the  patient  is  not  markedly  asthenic  or  feb- 
rile.    The  immediate  clinical  effects  of  tuberculin 


206  APPLIED  IMMUNOLOGY 

inoculations  are  ofttimes  distinctive  and  impressive. 
Briefly  summarized  they  are:  (1)  local,  manifested  by 
improvement  in  healing  tendencies  at  a  former  opera- 
tive site,  disappearance  of  inflammatory  signs,  of 
cough,  expectoration  and  tubercle  bacilli  from  the  spu- 
tum, although  the  last  is  very  persistent  and  may  never 
take  place;  (2)  general,  comprising  improvement  in 
appetite,  digestion,  strength,  weight,  disappearance  of 
fever,  etc.,  and  (3)  mental,  that  is,  general  morale. 

The  various  forms  of  tuberculosis  in  which  tuber- 
culin has  proved  useful,  if  not  curative,  comprise 
pulmonar}',  genito-urinary,  bones  and  joints,  intes- 
tinal, peritoneal,  laryngeal,  ocular,  mastoidal  and 
lymph-nodal. 

In  most  sanitaria,  tuberculin  is  routinely  employed 
in  selected  cases,  namely,  afebrile  or  mildly  febrile  pa- 
tients, when  an  incipient  infection  may  be  aborted  or 
the  course  of  a  chronic  loulmonary  process  favorably 
influenced.  In  1907,  Trudeau  stated  that  during  the 
past  15  years  the  post-discharge  mortality  of  patients 
from  the  Saranac  Sanitarium  was  18  to  25  per  cent, 
less  with  tuberculin-treated  than  non-treated  cases. 
Indeed  this  deduction  is  reasonable  in  view  of  the  fact 
that  tuberculin  cautiously  and  competently  adminis- 
tered is  the  best  expectorant  obtainable.  Recently, 
tuberculinum  purum,  "  T.  P.,"  has  been  extolled  in 
the  treatment  of  this  form  of  the  disease  (Fig.  18). 


208  APPLIED  IMMUNOLOGY 

The  forms  of  genito-urinary  tuberculosis  amenable 
to  tuberculin  include  hacillary  tuberculous  nephritis, 
where  both  kidneys  are  functionally  sufficient,  but  the 
primary  or  more  affected  kidney  is  indeterminable; 
bilateral,  functionally  insufficient,  hence  inoperable 
tuberculous  kidneys;  secondary  ureteritis  and  cystitis 
following  nephrectomy  for  tuberculosis  and  primary 
or  secondary  epididymitis,  prostatitis,  seminal  vesiculi- 
tis and  salpingitis.  Exact  discrimination  must  be 
exercised  in  the  latter  conditions  as  to  whether  the 
tuberculin  syringe  shall  precede  or  follow  the  scalpel. 

Perhaps  the  best  results  in  tuberculin  immuniza- 
tion have  occurred  in  the  treatment  of  chronic  osteitis 
and  arthritis.  Frequently  there  is  a  superimposed 
mixed  infection  in  these  cases,  calling  for  the  pre- 
liminary or  alternative  employment  of  autogenous 
bacterins ;  repeated  cultures  become  necessary  at  least 
biweekly,  to  identify  the  changeable  pyogenic  bacteria 
for  preparation  of  the  correct  bacterin.  We  have 
treated  many  such  cases  of  knee,  spinal,  and  hip- joint 
disease  with  excellent  results  (Fig.  19).  In  acute 
osteomyelitis  and  arthritis,  tuberculin  therapy  should 
be  condemned. 

Intestinal  and  peritoneal  tuberculosis  offers  little 
promise  for  tuberculin.  We  have  experienced  re- 
markable results  using  tuberculin  following  explora- 
tory laparotomy  revealing  extensive  nodular  tuber- 


JAN -I*  .t'^'Ht  .f 

It   -*  m  tf 


1 


J.C 

istered  oi 


TUBERCULIN  THERAPY  209 

culous  peritonitis,  and  we  have  seen  equally  bad  cases 
recover  without  tuberculin  after  simple  laparotomy. 

Tuberculous  laryngitis  is  nearly  always  associated 
with  pulmonary  involvement  and  tuberculin  has  at- 
tained but  indifferent  success,  although  a  few  ob- 
servers have  reported  excellent  results.  It  would  ap- 
pear that  the  association  of  local  treatment  is  a  most 
important  part  of  the  management  of  this  affection. 

Ocular  tuberculosis  includes  chronic  conjunctivitis, 
phlyctcenular  and  interstitial  keratitis,  iritis,  iridocycli- 
tis and  retinochoroiditis,  and  seems  to  be  a  field  wherein 
the  results  of  tuberculin  therapy  have  been  most 
happy.  Remarkable  results  have  been  obtained  with 
"  B.  E."  and  to  a  less  extent  with  "  T.  R."  Old 
tuberculin  seems  to  be  definitely  contra-indicated  in 
all  ocular  affections. 

Chronic  mastoiditis  and  otitis  media,  at  times,  have 
responded  brilliantly  to  tuberculin  immunization. 
Again,  when  the  inoculations  have  been  without  effect, 
operative  intervention  has  revealed  carious  bone. 

Tuberculin  therapy  in  chronic  cervical  lympha- 
denitis, in  the  absence  of  indicated  lymphadectomy, 
that  is,  when  the  nodes  have  undergone  marked  casea- 
tion or  hquefactive  necrosis,  and  particularly  follow- 
ing the  removal  of  diseased  tonsils  and  adenoids,  has 
enjoyed  considerable  success  and  should  be  employed 
in  the  future  more  extensively  than  has  been  the  cus- 

14 


210  APPLIED  IMMUNOLOGY 

torn  in  the  past.  The  patient's  general  health  im- 
proves immediately,  rapidly  and  markedly,  and  if  old 
sinuses  are  present  they  close  promptly.  In  our  ex- 
perience, equally  good  results  have  been  obtained  from 
"  T.  R.,"  "  B.  E.,"  "  O.  T."  and  "  T.  P."  The  re- 
sults of  treatment  by  tuberculin  in  this  form  of  tuber- 
culosis show  that  75  per  cent,  are  markedly  improved, 
and  that  40  per  cent,  of  these  will  be  cm^ed,  while  35 
per  cent,  will  demonstrate  only  slight  improvement, 
and  10  to  25  per  cent,  must  be  counted  as  failures. 
The  best  results  will  be  observed  in  children  under  the 
age  of  six. 


XVII 

PHAGOCYTOSIS 
By  phagocytosis  is  meant  a  property  of  the  leuco- 
cytes whereby  they  take  up  into  their  substance 
foreign  particles,  such  as  bacteria  (see  Frontispiece), 
pigment,  carbon  granules,  etc.,  thus  removing  them 
from  the  circulation.  This  power  belongs  pre- 
eminently to  the  polymorphonuclear  neutrophiles, 
though  phagocytosis  by  lymphocytes  is  occasionally 
observed.  This  phenomenon  was  discovered  by 
MetchnikofT,  who  regarded  it  as  the  principal  if  not 
the  sole  means  at  the  disposal  of  the  body  to  rid  itself 
of  disease-producing  germs.  According  to  his  teach- 
ing, phagocytosis  included  the  taking  up,  the  killing, 
and  digestion  of  living  bacteria,  as  well  as  absorption 
of  dead  bacteria  and  inert  materials.  Later  studies 
of  this  phenomenon  have  led  to  modification  of  this 
comprehensive  view  by  the  majority  of  observers.  It 
has,  for  example,  been  pointed  out  that  the  absorption 
of  live  bacteria  by  the  leucocytes  does  not  necessarily 
lead  to  their  destruction,  and  the  pus  of  gonorrhoea 
needs  only  to  be  cited  as  evidence.  In  gonorrhoea  the 
great  majority  of  the  gonococci  lie  within  the  leuco- 
cytes and,  far  from  undergoing  destruction,  rather 
tend  to  multiplication,  at  the  same  time  retaining  their 
highly  infectious  nature.     Experiments  with  other 

211 


212  APPLIED  IMMUNOLOGY 

varieties  of  bacteria  have  shown  that  they  also  may 
retain  their  vitahty  after  being  taken  up  by  the  leuco- 
cytes. On  the  other  hand,  it  has  been  shown  that  dead 
bacteria  are  taken  up  and  digested  by  the  leucocytes, 
and  that  insoluble  foreign  particles,  such  as  carbon, 
pigment,  etc.,  are  absorbed  and  deposited  in  parts  of 
the  body  where  they  will  do  no  harm.  It  would  ap- 
pear, therefore,  that  this  power  of  the  leucocytes, 
known  as  phagocytosis,  applies  to  the  taking  up  of  all 
foreign  particles,  irrespective  of  whether  they  are  liv- 
ing or  dead  bacteria,  pigment,  etc.,  thus  removing 
them  from  the  circulation,  but  in  itself  has  little  or 
nothing  to  do  with  destruction  of  the  life  of  bacteria. 
Phagocytic  action  is  to  be  distinguished  from  the 
bactericidal  action  of  the  leucocytes,  which  depends 
upon  separated  soluble  substances  and  does  not  take 
place  within  the  leucocytes  themselves. 

Phagocytosis,  therefore,  is  more  or  less  a  passive 
endeavor  to  remove  foreign  particles  from  the  circula- 
tion, independently  of  whether  they  are  living  or  dead, 
and  in  this  way  is  to  be  regarded  as  one  of  the  de- 
fensive forces  of  the  organism  against  disease.  The 
killing  and  destruction  of  living  bacteria  and  neutrali- 
zation of  their  toxins  are  carried  out  by  means  of  bac- 
teriolysins,  opsonins,  agglutinins,  and  antitoxins,  in 
the  soft  tissues  of  the  body,  processes  that  are  quite 
distinct  from  phagocytosis. 


XVIII 

RECOVERY  FROM  BACTERIAL  INFECTIONS 

In  the  combat  waged  against  disease,  the  body  is 
equipped  with  natural  defences  that  are  called  into 
play  by  the  entrance  of  bacteria.  In  addition,  each 
particular  form  of  infection  excites  the  production  of 
antibodies  that  are  specific  for  the  disease  in  question. 
By  means  of  these  non-specific  and  specific  defences, 
spontaneous  or  natural  recovery  is  brought  about. 

In  the  case  of  the  bacteria  that  produce  their  dele- 
terious effects  by  means  of  separated  soluble  toxins, 
for  example  the  diphtheria  and  tetanus  bacilli,  the 
body  defences  take  the  form  of  specific  antitoxic  sub- 
stances, which,  by  neutralizing  the  toxins,  cause  a 
secondary  destruction  of  the  infecting  organisms. 
Where  the  invading  bacteria  act  by  means  of  endo- 
toxins that  are  inseparably  bound  up  within  their 
protoplasm,  the  body  acts  directly  against  the  micro- 
organisms themselves  through  various  soluble  bac- 
tericidal substances  in  the  blood-fluid.  After  the  bac- 
teria have  been  killed,  their  bodies  are  taken  up  by 
the  leucocytes  (phagocytosis),  and  their  endotoxins 
neutralized  by  non-specific  oxidizing  substances  in  the 
leucocytes. 

Experimental  and  clinical  evidence  leads  to  the 
belief  that  there  are  in  the  blood  two  types  of  bac- 

213 


214  APPLIED  IMMUNOLOGY 

tericidal  substances,  (a)  leucocytic  bacteriocidin,  (b) 
humoral  bacteriocidin,  and  that  certain  varieties  of 
bacteria  are  destroyed  by  the  one,  and  other  varieties 
by  the  other.  These  two  types  of  bactericidal  sub- 
stances are  found  in  the  fluid  part  of  uncoagulated 
blood,  that  is,  in  the  plasma.  If,  however,  the  blood 
is  allowed  to  coagulate,  or  is  defibrinated,  only  the 
humoral  substance  is  found  in  the  serum.  From  this 
it  is  concluded  that  one  bactericidal  substance  is  de- 
rived from  the  leucocytes,  and  is  probably  identical 
with  fibrinogen,  while  the  serum  or  humoral  bacterio- 
cidin  does  not  come  from  the  leucocytes. 

It  has  been  found  that  streptococci  and  pneumo- 
cocci  are  killed  only  by  the  blood-plasma,  i,e,,  by  the 
leucocytic  substances;  these  organisms  multiply  in 
serum  alone.  On  the  other  hand,  typhoid  bacilli  are 
destroyed  by  serum  or  humoral  bacteriocidins.  In  this 
way  is  explained  the  clinical  fact  that  in  streptococcic 
and  allied  infections  there  is  a  marked  leucocytosis, 
while  in  typhoid  fever  the  leucocyte  count  remains 
normal. 

It  has  further  been  found  that  the  humoral  bac- 
tericidal substances  lose  their  activity  by  heating  the 
serum  to  55^  C,  but  that  they  can  be  reactivated  by  the 
addition  of  fresh  normal  serum;  in  other  words,  their 
action  depends  on  the  presence  of  complement.  On  the 
other  hand,  the  leucocytic  substances  are  not  inacti- 


BACTERLiL  INFECTIONS  215 

vated  by  heating  to  55°  C,  and  are  not  dependent  upon 
the  presence  of  complement  for  their  action.  It  is 
probable  that  the  humoral  bactericidal  substances  are 
specific,  each  one  acting  against  a  certain  microorgan- 
ism, while  the  leucocytic  substance  is  non-specific. 

Among  the  natural  defences  of  the  body  also  are 
substances  in  the  blood  known  as  opsonins,  which  have 
the  property  of  preparing  bacteria  for  phagocytosis 
by  the  leucocytes.  Whether  these  are  distinct  soluble 
substances  or  only  a  property  of  the  serum  is  not  de- 
termined; it  is  quite  likely  opsonins  are  a  modified 
form  of  the  bactericidal  substances  in  the  blood-fluid. 

From  the  foregoing  discussion  and  from  previous 
chapters,  we  see  that  the  principal  defences  of  the 
body  against  the  deleterious  effects  of  bacterial  infec- 
tion include  antitoxins,  agglutinins,  bacteriocidins 
(bacteriolysins),  opsonins  and  phagocytosis,  each  of 
these  playing  a  distinct  part  in  the  spontaneous 
recovery  of  the  organism  from  disease.  By  a  knowl- 
edge of  the  way  in  which  different  varieties  of  bac- 
teria produce  their  deleterious  effects,  and  the  re- 
sources present  in  the  body  for  meeting  the  attack  of 
the  different  species,  we  are  enabled  to  assist  nature  in 
combating  different  forms  of  infection  by  artificial 
stimulation  of  these  natural  resources.  This  is 
brought  about  by  the  various  general  therapeutic 
measures,  and  in  a  specific  manner  by  bacterin  therapy. 


XIX 

BACTERIAL  INOCULATION 

PRINCIPLES  UNDERLYING  INOCULATION  THERAPY— PREP- 
ARATION OF  BACTERINS— AUTOGENOUS  VERSUS  HET- 
EROGENEOUS PREPARATIONS— CLINICAL  SYMPTOMS 
VERSUS  OPSONIC  INDEX  IN  CONTROL  OF  TREATMENT 

Principles  Underlying  Therapeutic  Inoculation. 
— It  has  been  elsewhere  stated  that  bacterial  inocula- 
tions operate  by  stimulation  of  tissue  cells,  after  sub- 
cutaneous, intramuscular,  or  intravenous  injection, 
to  the  production  of  specific  antibodies,  be  they  named 
agglutinins,  bacteriolysins,  opsonins,  or  what  not. 
From  their  source,  they  are  taken  up  by  the  Ij^mph 
iand  blood-serum  and  distributed  throughout  the  body. 
In  the  morbid  process  their  action  is  to  sensitize  the 
invading  bacterin,  whereby  they  become  more  readily 
devoured  by  certain  leucocytes,  hence  phagocytosis  is 
promoted,  or  the  antibodies  by  virtue  of  their  lytic 
properties  attack  their  specific  bacteria,  resulting  in 
their  disintegration  (bacteriolysins),  and  tissue  re- 
pair is  favorably  influenced. 

A  patient  smitten  by  an  acute  infection  is  the 
victim  of  an  inoculation  of  living  bacteria  (antigen). 
If  his  resistance  be  poor  and  the  virulence  of  the  in- 
vading microbe  be  great,  he  is  prone  to  die;  if  his  re- 
sistance be  par  or  better,  and  the  bacterium  relatively 

216 


BACTERL\L  INOCULATION  217 

avirulent  or  the  dose  small,  recovery  is  to  be  expected : 
on  the  other  hand,  although  his  vital  resistance  be 
exceptionally  good,  a  severe  virulent  infection  will 
probably  result  fatally.  In  any  case  it  is  obvious  that 
the  size  of  the  living  bacterial  inoculation  is  too  large 
— also  more  potent  than  the  commonly  employed 
maximum  therapeutic  inoculation  of  dead  bacteria — 
for  the  welfare  of  the  patient  and  he  is  temporarily 
thrust  into  the  so-called  "  negative  phase."  Thera- 
peutically, therefore,  bacterin  therapy  is  indicated 
only  in  chronic  or  possibly  also  subacute  localized  in- 
fections. It  is  impossible  to  conceive  by  any  stretch 
of  the  imagination  the  rationale  or  at  least  the  necessity 
of  their  employment  in  acute  or  diffused  infections 
in  which  the  human  organism  is  already  overloaded 
with  the  products  of  a  living  antigen,  or  indeed  its 
defences  actually  demolished  and  its  system  over- 
whelmed by  the  invading  microbes;  namely,  such 
states  as  bactersemia  or  septicaemia.  Indeed,  bacterial 
inoculations  in  such  conditions  not  only  add  insult  to 
injury  but  may  be  the  determining  forces  for  death 
instead  of  prolonged  recovery.  Administered  in  acute 
localized  aiFections,  as  a  rule  unwisely  perhaps,  and 
certainly  so  unless  the  indications  of  the  opsonic  index 
are  followed,  either  of  two  possible  results  may  be  ex- 
pected. If  the  bodily  resources  are  barely  able  to 
cope  with  the  situation,  and  the  local  infection  be  very 


218  APPLIED  IMMUNOLOGY 

active,  additional  injection  of  antigen  will  turn  the 
tide  of  battle  against  the  individual  and  an  otherwise 
acute  course  be  converted  into  a  chronic  or  vital  issue. 
On  the  other  hand,  if  the  individual's  defences  are 
great  and  his  infection  slight,  an  inoculation  of  bac- 
terin  may  possibly  increase  his  specific  antibodies, 
thereby  enabling  him  to  abort  or  at  least  shorten  the 
attack  of  his  local  infection.  In  the  latter  case,  the 
probability  is  that  the  patient  if  left  to  himself  will 
readily  conquer  his  infection  in  due  time.  This  dis- 
crimination, in  view  of  the  danger  involved,  between 
leaving  Nature  to  herself  and  intervention  by  bacterin 
therapy,  can  be  made  only  by  recourse  to  determina- 
tions of  the  opsonic  index.  As  a  rule,  these  acutely 
localized,  particular^  if  severe,  and  bacter^emic  con- 
ditions constitute  realms  for  the  wiser  and  more  logical 
practice  of  serum  therapy  or  passive  immunization. 
Consensus  of  opinion  generally  supports  the  belief 
that  the  bacteria  best  adapted  to  bacterin  therapy  or 
active  immunization  are  those  containing  the  poison- 
ous substances  within  themselves,  the  so-called  "  endo- 
toxins," typified  by  the  staphylococci  and  strepto- 
cocci. Such  bacteria  do  not  liberate  their  endotoxins 
until  disintegration  or  death  of  their  organism  occurs. 
On  the  other  hand,  diseases  caused  by  the  "  exoteric  " 
or  toxin-producing  microorganisms,  typified  by  diph- 


o 

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P  o 
3  =r 
r  P 
3  3 
^^' 


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o  o 

3  C 


0-2. 


n  o 
0-  . 


S  ° 
P  3 

c  S. 


3  D. 

(R  p 


BACTERIAL  INOCULATION  219 

theria  and  tetanus,  are  more  appropriately  treated  by 
immune  sera  or  antitoxins. 

Preparation  of  Bacterins. — The  earliest,  simplest 
and  commonest  method  of  bacterin  preparation  con- 
sists in  washing  off  the  appropriate  media  twenty- 
four-hour — with  certain  bacteria  the  time  is  much 
longer — pure  cultures  of  the  bacterium,  using  a  plati- 
num spatula.  The  growth  is  suspended  in  normal 
saline  solution,  containing,  if  desired,  0.25  to  0.5  per 
cent,  phenol.  After  shaking  for  one-half  hour  in  a 
mechanical  shaker  (Fig.  20),  the  suspension  is  stand- 
ardized to  contain  from  100,000,000  to  1,000,000,- 
000  bacteria  per  cubic  centimetre.  This  is  best 
accomplished  by  the  hsemocytometer  method,  first 
employed  by  one  of  the  authors  (Thomas)  in  1906. 
The  bacterial  suspension  is  drawn  up  in  an  ordinary 
red  blood-cell  counting  pipette  to  the  mark  0.5,  fol- 
lowed by  freshly  filtered  carbol-thionin,  Leishman's 
or  Jenner's  stain,  sufficient  to  stain  the  bacteria.  After 
mild  agitation  for  two  or  three  minutes  the  bulb  is 
filled  to  the  101  mark,  and  after  thorough  dissemina- 
tion of  the  bacteria  a  drop  of  the  pipette  content  is 
placed  on  the  hsemocytometer  stage  and  the  micro- 
organisms are  comited  precisely  as  are  red  blood-cells, 
determining  the  number  per  cubic  centimetre.  It  is 
advisable  to  use  a  cover-slip  not  over  0.13  mm.  thick; 
otherwise  there  may  be  interference  with  the  oil-im- 


220  APPLIED  IMMUNOLOGY 

mersion  objective.  Recently,  Glynn,  Rees,  Powell 
and  Cox  have  recommended  a  special  counting-cham- 
ber 0.02  mm.  deep,  instead  of  the  common  0.1  mm. 
depth,  with  which  a  heavier,  0.18  mm.  thick,  cover- 
slip,  suitable  for  all  achromatic  oil-immersion  lenses, 
even  with  a  free  working  distance  as  low  as  0.09  mm., 
can  be  used.  It  is  claimed  that  with  the  shallower 
chamber  the  bacteria  settle  to  the  bottom  in  fifteen 
minutes,  while  with  the  deeper  they  are  still  in  motion 
after  one-half  hour,  therefore,  with  the  special  hsemo- 
cytometer  they  are  more  easily  enumerated  for  focal 
reasons,  their  definition  is  clearer  cut  and  the  heavier 
cover-slip  is  more  durable.  Any  hsemocytometer 
method  is  more  accurate  than  Wright's  method,  which 
underestimates  the  number  of  bacteria  in  suspension 
and  possesses  an  average  and  maximum  error  three 
times  as  great  as  the  h^emocytometer. 

Wright's  method  consists  in  mixing  a  unit  volume 
of  the  bacterial  suspension  with  an  equal  volume  of 
normal  blood  known  to  contain  approximately  5,000,- 
000  erythrocytes  to  the  cubic  millimetre  or  5,000,000,- 
000  to  the  cubic  centimetre.  This  is  diluted  by  the 
addition  of  six  or  seven  volumes  of  0.15  per  cent, 
sodium  citrate  solution  to  prevent  clotting.  After 
thorough  mixing,  fairly  thick  smears  are  made  on 
glass  slides  and  stained  preparatory  to  the  count. 
Fields  throughout  the  smear  are  counted  for  the  sake 


BACTERIAL  INOCULATION  221 

of  accuracy  and  the  proportionate  number  of  bacteria 
to  the  red  blood-cells  counted,  determined  as  follows : 
If  in  fields  containing  1000  red  blood-cells  500  bac- 
teria are  counted  we  have  the  proportion 

1000  :  500  ::  5,000,000,000  :  x. 

X  =z  2,500,000,000,  or  the  number  of  bacteria  per  c.c.  of  suspension. 

This  suspension  is  obviously  too  concentrated  and 
may  be  conveniently  diluted  as  follows:  If  it  is  de- 
sired to  prepare  the  bacterin  in  10  c.c.  containers,  so 
that  each  cubic  centimetre  shall  contain  400,000,000 
bacteria,  we  have  the  formula 

400,000,000  X  10 


2,500,000,000 


=^  x  =  1.6  c.c. 


Therefore  to  1.6  c.c.  of  the  original  suspension 
must  be  added  8.4  c.c.  of  the  diluting  fluid,  phenol-salt 
solution.  The  bacterin  is  then  sterilized  by  submer- 
sion in  a  water-bath  for  one-half  to  one  hour  at  the 
thermic  death-point  of  the  particular  bacterium,  usu- 
ally about  60°  C.  After  removal  from  the  bath,  a 
drop  of  the  bacterin  is  cultured  for  a  day  or  two  in 
order  to  test  its  sterility  before  employment  thera- 
peutically. Other  methods  of  sterilization  occasion- 
ally utilized  include  chemical,  cold  and  maceration  by 
prolonged  shaking. 

Preparation  of  Bacterins  by  Autolysis. — Certain 
investigators  have  attempted  to  improve  bacterins 
and  enhance  their  immunizing  properties  by  subject- 


222  APPLIED  IMMUNOLOGY 

ing  the  bacteria  to  a  process  of  autolysis,  on  the  as- 
sumption that  the  autolysis  contains  toxic  or  anti- 
opsonic  substances,  and  that  the  antigenic  part  of  the 
bacterin  depends  upon  the  bacteria  themselves  minus 
their  extractives.  This  disintegration  of  the  bacteria 
is  accomplished  by  treating  them  with  salt  solution, 
alcohol,  ether,  chloroform,  or  xylol,  followed  by  filtra- 
tion and  suspension  of  the  residue  in  normal  saline. 

Serohacterins. — Recently,  advantage  has  been 
taken  of  Besredka  s  assertion,  founded  upon  the  dem- 
onstration of  Ehrlich  and  Morgenroth,  that  inocula- 
tions with  sensitized  bacteria  supersede  in  eiFective- 
ness,  rapidity  of  action  and  harmlessness  ordinary 
bacterins  or  bacterial  vaccines.  Such  sensitized  bac- 
terins  or  "serohacterins"  are  prepared  by  treating  bac- 
teria with  their  specific  immune  serum,  by  which 
process  the  bacteria  become  inseparably  joined  with 
their  antibodies,  thereby  in  a  state,  immediately  on  in- 
jection, prepared  to  be  acted  upon  by  the  complement 
of  the  patient's  blood.  Thus  the  usual  delay  of  a  week 
or  more,  required  for  the  patient  to  form  his  own  anti- 
bodies— incidentally  also  the  negative  phase  and  local 
injectional  reaction — using  the  ordinary  bacterins,  is 
avoided,  and  immunity  is  secured  rapidly  and  inten- 
sively in  twenty-four  to  forty-eight  hours.  Finally, 
by  virtue  of  the  absence  of  "  negative  phases,"  the  in- 
oculations may  be  repeated  every  day  or  two,  thereby 


BACTERIAL  INOCULATION 


223 


markedly  shortening  the  course  of  treatment.  Should 
sensitized  bacterins  fulfil  their  promise,  they  will  mark 
the  greatest  advance  thus  far  in  the  history  of  bacterin 
therapy. 

Bacterins,  like  tuberculins,  are  best  stored  in  a 
dry,  cool,  dark  place.  As  a  matter  of  convenience  they 
are  put  up  in  hermetically  sealed  ampoules  or  small 


Fig.  21. — Various  forms  of  containers  for  storage  of  bacterins. 

bottles  or  vials  fitted  vi^ith  ground  glass  stoppers  or 
rubber  caps  (Fig,  21).  The  standardized  strength 
of  the  bacterin,  that  is,  the  number  of  bacteria  per 
cubic  centimetre,  also  its  date  of  preparation,  should 
be  inscribed  on  the  container.  The  preservative,  phe- 
nol, usually  added  to  the  diluting  fluid,  insures  against 
contamination  or  bacterial  growth  in  the  preparation. 
It  should  be  remembered  that  the  bacterin  may  ap- 


224  APPLIED  IMMUNOLOGY 

pear  perfectly  clear  after  a  certain  time  owing  to 
digestion  of  the  previously  discernible  microorganisms. 
On  the  other  hand,  it  often  happens  that  the  bac- 
terin  becomes  clouded  or  a  precipitate  forms,  due  to 
the  chemical  action  of  the  contained  phenol.  Such 
bacterins  should  be  immediately  discarded.  As  a  rule, 
bacterial  suspensions  in  trikresol-saline  solution  will 
keep  for  months.  It  is  alleged  that  typhoid  bacterin 
becomes  inert  after  three  months. 

Autogenous  versus  Stock  Bacterins, — By  an  autog- 
enous or  personal  bacterin  is  meant  one  that  is  pre- 
pared from  the  particular  bacterium  or  strain  of  bac- 
terium cultured  from  the  infected  patient.  A  stock 
or  laboratory  bacterin  is  understood  to  mean  a  prep- 
aration in  which  the  bacterium  has  been  isolated  from 
another  patient,  who  has  suffered  from  a  similar  in- 
fection. In  view  of  the  diversity  of  the  strains  of 
many  bacteria,  notably  the  streptococcus,  colon  ba- 
cillus, etc.,  also  of  the  fact  that  not  a  few  diseases  are 
caused  by  different  bacteria,  namely,  abscess,  acne, 
etc.,  it  is  natural  that  the  preferable  and  most  scientific 
procedure  is  to  employ  an  autogenous  bacterin  when- 
ever possible.  The  authors  have  often  successfully 
treated  cases  with  an  autogenous  preparation  after 
stock  bacterins  have  resulted  in  failure.  Not  infre- 
quently it  becomes  expedient  to  use  a  stock  prepara- 
tion, at  least  while  the  autogenous  bacterin  is  being 


BACTERIAL  INOCULATION  225 

prepared.  Under  such  circumstances,  notably  in 
gonorrhoea!  and  tuberculous  affections,  it  is  impera- 
tive that  the  infecting  germ  be  accurately  determined. 
Moreover,  the  bacterin  should  be  polyvalent,  that  is, 
composed  of  as  many  different  strains  of  the  bac- 
terium as  may  be  obtainable,  and  in  view  of  the  reports 
of  Besredka,  Broughton-Alcock,  Ardin-Deltiel, 
Negre,  Raynaud,  Gordon,  Boinet,  Cruveillier  and 
others,  it  would  appear  advisable  to  employ  so-called 
sensitized  bacterins. 

Clinical  Symptoms  versus  Opsonic  Indeos  in  Con- 
trol of  Treatment, — Regulation  of  the  size  of  doses  of 
bacterins  and  the  spacing  of  the  intervals  between  in- 
oculations is  possible  by  observations  of  the  clinical 
symptoms,  subjective  and  objective,  and  by  determi- 
nations of  the  opsonic  index.  Both  methods  involve 
special  knowledge  and  are  not  to  be  undertaken 
lightly.  The  former  entails  the  closest  kind  of  ob- 
servation respecting  local,  focal  and  general  responses 
( see  Chapters  XVI  and  XXI ) .  Opsonins  are  not  the 
only  antibodies  formed  after  inoculation  of  an  ani- 
mal, hence  the  opsonic  index,  theoretically,  cannot 
measure  the  full  degree  of  immunity.^  Practically, 
the  opsonic  index  in  the  majority  of  cases  runs  par- 

*  There  is  no  such  thing  as  absolute  immunity,  however,  a  fact  pointed 
out  by  Pasteur  years  ago,  for  an  animal  will  contract  disease,  irrespective 
of  the  degree  of  immunization,  provided  the  dose  of  infective  material  is 
suflBciently  large  and  virulent. 

15 


226  APPLIED  IMMUNOLOGY 

allel  with  the  cHnical  symptomatology,  and  although 
in  many  cases  it  is  unnecessary  to  employ  the  index 
as  a  guide,  there  are  many  cases  in  which  dependence 
upon  it  is  absolutely  essential  to  attain  the  greatest 
success.  To  ignore  the  index  absolutely  in  all  cases 
will  invite  disaster.  The  authors  feel,  after  an  ex- 
perience of  many  years,  that  the  most  brilliant  results 
— often  in  the  most  difficult  cases — have  attended 
bacterin  therapy,  wherein  the  opsonic  index  was  asso- 
ciated in  the  management  of  the  case;  although  they 
are  inclined  to  attach  primary  importance  to  the  clini- 
cal symptomatology,  properly  interpreted,  and  to 
relegate  the  index  to  second  place.  For  instance,  in 
the  treatment  of  acne  or  recurrent  furunculosis,  we 
are  not  infrequently  at  a  loss,  after  the  disappearance 
of  the  present  lesions,  to  say  whether  or  not  the  process 
of  immunization  has  been  carried  far  enough  to  insure 
no  recurrence,  or  in  certain  other  affections,  charac- 
terized by  a  high  fluctuating  temperature  and  tox- 
aemia, or  in  deep-seated  lesions,  such  as  pyelitis,  pye- 
lonephritis, cystitis,  etc.,  the  value  of  the  index  in 
controlling  dosage  and,  in  the  case  of  mixed  infec- 
tions, in  selecting  the  needed  bacterin,  is  not  incon- 
siderable. The  treatment  of  this  class  of  conditions 
lies  without  the  sphere  of  the  general  practitioner  and 
he  would  do  well  to  refer  such  cases  to  those  more 
particularly  versed  in  the  application  of  bacterial  in- 


BACTERLVL  INOCULATION  227 

oculations.  On  the  other  hand,  physicians  generally, 
at  the  present  day,  owing  largely  to  the  energy  of 
pharmaceutical  firms  marketing  stock  bacterins,  em- 
ploy bacterins  controlled  or  miscontrolled  by  the  clini- 
cal symptoms  in  preference  to  the  opsonic  index. 

It  is  utterly  out  of  the  question  for  the  average 
general  practitioner  to  master  the  bacteriology  and 
laboratory  technic  required  for  the  reliable  determina- 
tion of  the  opsonic  index,  and  experience  has  already 
demonstrated  that  in  the  majority  of  patients  thus 
far  subjected  to  bacterial  immunization  the  clinical 
symptomatology  has  admirably  sufficed  to  control 
the  inoculations.  Consequently,  bacterin  therapy,  in 
order  to  enjoy  the  popularity  which  is  its  due,  must  be 
governed,  within  limitations,  by  the  subjective  and  ob- 
jective symptoms  and  signs,  observing  the  former  and 
avoiding  the  latter. 


XX 

THE  OPSONIC  INDEX 

DEFINITION  OF  OPSONINS  AND  THE  OPSONIC  INDEX— 
TECHNIC  OF  DETERMINATION  OF  THE  OPSONIC  INDEX 
—INTERPRETATION,  VALUE  AND  LIMITATIONS  OF  THE 
OPSONIC  INDEX 

Denys  and  Leclef,  Leisliman,  Wright  and  Doug- 
las and  others  have  demonstrated  that  the  real  activity 
of  phagocytes  depends  upon  a  specific  substance — 
the  exact  chemical  nature  of  which  has  been  unde- 
termined— existing  in  the  serum  of  the  blood.  Irre- 
spective of  a  certain  amount  of  spontaneous  phag- 
ocytosis, which  has  been  shown  may  take  place,  Sir 
Almroth  E.  Wright,  of  St.  Mary's  Hospital,  London, 
has  definitely  proved  that  the  subcutaneous  injection 
of  dead  bacteria  is  capable  of  the  production  of  spe- 
cific antibody,  which  taken  up  by  the  blood-serum 
markedly  augments  the  ability  of  the  phagocyting 
leucocytes  to  devour  invading  pathogenic  bacteria. 
To  this  specific  substance,  capable  of  preparing  bac- 
teria for  ingestion,  Wright  assigned  the  name  "  opso- 
nin," from  the  Greek  verb  bi^ovid,  I  prepare  food 
for.  Opsonins,  therefore,  are  those  specific  substances 
or  antibodies,  not  yet  isolated,  in  the  blood-serum, 
possessed  of  the  property  to  sensitize  or  prepare  bac- 
teria for  phagocytosis.    Opsonins  are  bacteriotropins 

228 


THE  OPSONIC  INDEX  229 

and  should  not  be  confounded  with  bacteriolysins, 
another  type  of  antibody.  Moreover,  it  has  been 
shown  that  opsonins  are  divisible  into  thermolabile 
(the  normal  opsonin  of  Wright)  and  thermostable 
( immune  opsonin ) .  The  former  are  readily  destroyed 
at  a  temperature  of  dQ""  C,  the  latter  retain  their  bac- 
teriotropic  property  in  spite  of  this  degree  of  heat. 

By  virtue  of  the  assiduous  labors  of  Wright  and 
his  co-workers  in  evolving  a  technic  (determination  of 
the  opsonic  index)  to  measure  phagocytosis  (degree 
of  immunization),  therapy  by  bacterial  inoculations 
was  revived  and  popularized  to  an  unprecedented  ex- 
tent and  has  led  to  the  application  of  active  immuniza- 
tion of  far-reaching  consequences,  and  it  is  for  this 
reason  that  medicine  owes  Wright  a  perennial  debt. 

The  opsonic  index  may  he  described  as  the  measure 
of  the  ratio  of  the  phagocytic  activity  of  neutral  or 
washed  leucocytes  in  the  patient's  serum  for  given 
bacteria,  as  compared  with  those  in  a  normal  or  con- 
trol serum.  Inasmuch  as  a  neutral  phagocyte  will  in- 
gest the  same  number  of  bacteria,  provided  the  two 
sera  possess  identical  qualities,  the  normal  or  base  line 
is  arbitrarily  taken  as  one.  If,  in  the  ease  of  the  pa- 
tient's serum,  it  is  found  that  100  leucocytes  contain 
900  bacteria,  while  the  same  number  of  leucocytes 
treated  with  the  control  serum  contain  1000  bacteria, 


230  APPLIED  IlViMUNOLOGY 

we  have  the  proportion  of  900  :  1000  ::  tr  :  1,  in  which 
00,  the  opsonic  index,  equals  0.9. 

Teclinic  of  Determination  of  the  Opsonic  Index. 
— The  technic  of  the  opsonic  index  is  a  rather  intricate 
laboratory  procedure,  which,  to  be  reliable  or  trust- 
worthy at  all,  involves  much  practice  in  mastering  the 
details  as  stipulated  by  Wright.  It  demands  that  the 
opsonist  shall  be  a  thorough  bacteriologist  as  well  as 
an  experienced  laboratory  worker.  Briefly,  the  pro- 
cedure may  be  described  as  follows: 

Material  and  apparatus  required, 

(a)  Pure  culture  of  infective  bacterium. 

(b)  Specimen  of  serum  of  patient's  blood. 

(c)  Specimen  of  serum  of  normal  or  control 
blood. 

(d)  Normal  washed  leucocytes. 

(e)  Centrifuge,  preferably  electric. 

(f)  Thermostat  or  opsonizer. 

(g)  Centrifuge  or  special  test-tubes. 

(h)  Teat  and  capillary  pipettes,  glass  tubing, 
slides,  absolute  alcohol,  stains,  etc. 

(i)   Microscope. 

A  few  drops  of  the  patient's  blood  and  of  the  nor- 
mal or  control  blood  are  collected  in  two  glass  tubes 
drawn  out  on  either  end  to  capillary  size  (Fig.  22). 
The  straight  or  distal  end,  free  of  blood,  is  sealed  in  a 
flame,  and  in  a  few  minutes,  after  the  tube  cools,  dur- 


Fig.  23. 


Fig.  24. 


^^^ 


¥ 


Fig.  25. — Electric  centrifuge.  This  instrument  is  particularly  well  adapted  for 
the  preparation  of  washed  leucocytes  by  virtue  of  the  easy  manner  in  which  it  loses 
speed  when  the  current  is  broken,  thereby  avoiding  disturbance  of  the  layer  of 
"leucocytic  cream." 


baciena\ 
5u5pen5'\onv 


Fi 


w 


\ 


Fig.   26. — \'arious  ingredients  neoes-sary  for  the  determination  of  the  opsonir  index 


THE  OPSONIC  INDEX 


231 


ing  which  time  the  blood  coagulates  and  is  spontane- 
ously drawn  toward  the  sealed  end,  the  curved  or 
inlet  end  may  also  be  sealed.  The  serum  begins  to  lose 
its  opsonic  content  in  six  hours  and  should  not  be 
utilized  for  index  determination  after  this  time.  Ten 
to  fifteen  drops  of  normal  blood 
are  then  allowed  to  fall  from  the 
finger-tip  into  a  test-tube  of  normal 
salt  solution  containing  1.5  per  cent, 
sodium  citrate  (Fig.  23).  The 
citrate  will  prevent  the  clotting  of  the 
blood,  and  the  corpuscles  are  thor- 
oughly washed  by  centrifugation  two 
or  three  times  in  normal  saline  (Fig. 
25 ) ,  to  render  them  neutral  and  abso- 
lutely free  from  serum.  The  white 
blood-corpuscles,  being  lighter  than 
the  red,  will  overlie  the  latter  as  a 
grayish  layer  of  "  leucocytic  cream  " 
(Fig.  24),  whereupon  they  may  be 
picked  up  by  a  pipette  and  trans- 
ferred to  another  receptacle  (Fig. 
A  six-  to  twenty-four-hour  culture  of  the 
given  bacterium  is  washed  off  in  normal  salt  solu- 
tion (Fig.  27),  then  centrifuged  to  throw  down 
all  bacterial  clumps.  The  supernatant  suspension 
should  contain  only  individual  bacteria  in  not  too 


1/ 


Fio.   22. 


-Capillary 
glass  capsules  for  the 
collection  of  specimens 
of  blood.  A,  simple 
straight;    B,   Wright's. 


26 


232 


APPLIED  IMMUNOLOGY 


great  concentration.  The  best  strength  would  ap- 
pear to  be  that  rendering  a  phagocytosis  averag- 
ing about  four  bacteria  pro  leucocyte.  Two  opsoniz- 
ing capillary  pipettes,  P  (patient)  and  C  (control), 
are  prepared  so  that  the  lumina  are  about  the  diame- 
ter of  a  large  sized  hat-pin.    A  distance  of  one-half 


Fig.  27. — Washing  the  culture  of  the  given  bacterium  from  the  culture  medium,  using 
the  platinum  loop,  in  order  to  obtain  the  bacterial  suspension. 

or  three-quarters  of  an  inch  is  marked  off  on  the  ends 
of  these  two  pipettes  (Fig.  28, 1  and  II) .  By  the  use 
of  a  small  rubber  bulb,  equal  quantities  of  patient's 
serum,  bacterial  suspension  and  washed  leucocytes, 
permitting  a  small  bubble  of  air  to  separate  each,  are 
drawn  up  and  mixed  by  skilful  thumb  and  finger 
gymnastics,  in  the  pipette  designated  P.    In  the  same 


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Fig.  29. — Opsonizer  or  thermostat.  This  incubator  i.s  equipped  with  an  automatic 
heating  device  and  permits  of  the  ready  manipulation  of  pipettes  introduced  through 
the  side  port-holes. 


THE  OPSONIC  INDEX 


233 


manner  equal  quantities  of  control  serum,  bacterial 
suspension  and  washed  leucocytes  are  taken  up  and 
mixed  in  pipette  C  (Fig.  28,  III  and  IV).  The 
tips  are  sealed  in  a  flame  and  the  two  pipettes  incu- 
bated for  a  few  minutes  in  a  thermostat  or  opsonizer 
at  37°  C.  (Fig.  29).    Care  should  be  taken  to  agitate 

the  contents  every  five  min- 
utes during  incubation, 
otherwise  the  corpuscles 
will  gravitate  and  a  per- 
fect admixture  of  serum, 
bacteria  and  phagocytes 
will  not  be  obtained.  At 
the  conclusion  usually  of 
fifteen  minutes,  smears  are 
made  of  the  contents  of  the 
pipettes  on  cover-slips  by 
the  usual  method  or  glass 
slides,  employing  Kulm- 
hardt's  spreader  (Figs.  30 
and  31).  After  drying, 
the  smears  are  fixed  in  absolute  alcohol  and  stained 
with  freshly  filtered  carbolthionin  or  by  the  method 
of  Homer  Wright.  In  the  determination  of  the 
tuberculo-opsonic  index,  the  culture  of  tubercle 
bacilli  is  killed  by  fractional  sterilization  on  three 
successive  days  at  100°  C,  then  thoroughly  ground 


Fig.  30. — Illustrating  the  construction 
of  Kuhnhardt's  spreader.  A  shows  a 
glass  slide  properly  nicked  by  a  file;  B, 
the  slide  p.cpcrly  broken  in  a  slightly 
concave  line;  C,  the  broken  slide  with 
corners  clipper" ;  D  and  E,  the  perfected 
spreader  spliced  to  a  second  slide  with 
adhesive  plaster. 


234 


APPLIED  IMMUNOLOGY 


in  an  agate  mortar  and  suspended  in  0.85  per  cent, 
solution  of  sodium  chloride.  After  fixation  of  the 
smears  either  by  ethyl  or  methyl  alcohol  or  by  heat, 
the  usual  staining  method  comprising  carbol-fuchsin, 
nitric  acid  and  methylene  blue  for  tubercle  bacilli  is 
employed  (see  Frontispiece).     The  number  of  bac- 


FiG.  31. — Kuhnhardt'3  spreader  properly  held  at  the  right  angle  for  the  preparation  of 
a  satiafactory  smear. 

teria  contained  in  no  fewer  than  one  hundred  phag- 
ocytes should  be  counted  and  the  index  determined  as 
noted  above;  or,  in  accordance  with  the  method  of 
Simon,  the  ratio  of  the  number  of  phagocji:ing  leuco- 
cytes may  be  determined.  The  latter  method  is 
simpler,  less  exacting  on  vision  and  possibly  just  as 
reliable  as  the  method  of  Wright. 


THE  OPSONIC  INDEX  235 

Interpretation,  Value  and  Limitations  of  the 
Opsonic  Index, — Wright  contends  that  the  opsonic 
index,  properly  done  and  interpreted,  is  a  reliable  ex- 
pression of  an  animal's  resistance  against  infection. 
The  effect  of  a  bacterial  inoculation,  after  a  transi- 
tory drop  in  the  opsonic  index,  is  an  increase  in  the 
degree  of  immunity  of  the  inoculated  animal  against 
the  disease  caused  by  the  given  bacterium.  The  pri- 
mary drop  or  so-called  *'  negative  phase,"  presumably 
due  to  the  absorption  of  preformed  opsonins  by  the 
injected  bacteria  or  to  the  systemic  effect  incident  to 
the  cellular  stimulation  of  the  organism  for  the  pro- 
duction of  specific  antibodies,  may  be  of  very  short  or 
prolonged  duration,  and  if  of  any  consequence  is 
characterized  by  the  clinical  phenomena,  general,  focal 
and  local,  described  in  the  following  chapter.  In  a 
few  hours,  after  a  proper  sized  dose,  the  opsonic  index 
rises  in  the  scale.  This  ascent  is  termed  the  "  flow  " 
and  may  fail  to  reach  the  normal  or  soar  well  above  it. 
Since  the  effect  is  but  transient,  the  index  will  slowly 
fall  and  this  descent  is  called  the  "  backflow "  or 
"  ebb  "  or  "  negative  phase,"  occasionally  following 
an  inoculation,  if  the  indices  are  determined  suffi- 
ciently often,  the  curve  will  exhibit  a  mild  "  initial 
rise  "  preceding  the  "  ebb  "  or  "  negative  phase,"  be- 
fore the  real  "  flow "  or  "  positive  phase  "  sets  in 


236 


APPLIED  IMMUNOLOGY 


( Fig.  32 ) .  In  the  conduct  of  therapeutic  inocula- 
tions it  should  be  the  purpose  of  the  immunologist  to 
repeat  the  inoculation  in  the  "  positive  phase  "  or  as 
the  "  ebb  "  begins.  Extremely  large  inoculations  or 
reinoculations  while  the  index  is  in  the  "  negative 


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t!                                             ^                   -       -^i           - 

i?                                    V^            -  ,-•'   t 

\l                                       "u5              -^ 

1^     ~                     Z                       S'            ^            ? 

M  -                      ^                     ±^,-^               : 

«         ♦          "  "^             4  ^i^  - 

Ji           B                 ^      '       lij:3^'^C__ 

^             \           ^        i        .^M.        t    -      ^  - 

\l  ii.«^"  t       i     \,2s^:^    __  .  (L    _  ._          _  _ 

w  «•« — -^  -  -I      -?T:i:           tl^^ 

^       i         /~  ^'  t5t^              ^  t 

^          li.^  t'^         l\~-            -  ^i             ,      - 

«I                    li:                    2    J 

A  —     ^'2              ~     J                                      ■       - 

^      e=^2''             o                               -            o 

«  — i      -s             -1                 .1      __    _  ±    _ 

Fig.  32. — Analysis  of  curve  of  opsonic  indices. 

phase  "  result  in  an  exaggeration  and  prolongation  of 
the  "  negative  phase  "  which  handicap  the  patient's 
recuperative  powers  and  may  suffice  to  turn  the  tide 
of  battle  against  him.  Inoculations  repeated  at  the 
correct  time  will  still  further  raise  the  opsonic  index 
above  the  normal,  until,  as  Nature  resumes  her  work, 


THE  OPSONIC  INDEX  237 

the  "  ebb "  is  less  conspicuous  and  the  "  positive 
phase  "  becomes  sustained.  At  this  time  the  patient 
is  said  to  be  on  the  "  high  tide  of  immunity  "  and 
further  inoculations  are  contra-indicated.  This  is  ob- 
served in  connection  with  prophylactic  inoculations 
and  where  recovery  is  apparent.  Wright's  original 
rule  was  to  take  the  opsonic  index  daily  and  be  guided 
exclusively  by  its  indications ;  at  present,  although  the 
value  of  the  index  is  not  belittled,  the  clinical  course 
of  the  case  is  utilized  as  an  important  guide  in  treat- 
ment. Inoculations  are  not  repeated  until  positive 
phase  symptoms  are  no  longer  apparent,  or  in  doubt- 
ful cases  determination  of  the  index  demonstrates  a 
fall  in  opsonins.  So  long  as  there  occurs  any  benefi- 
cial response  whatever  from  a  bacterial  inoculation, 
its  size,  whether  the  "  minimum  effective "  or  the 
"  medium  or  average,"  is  not  increased. 

The  "  ideal  curve  "  consists  of  a  "  negative  phase  '' 
of  twelve  or  twenty-four  hours,  followed  by  a  "  posi- 
tive phase  "  lasting  from  three  to  fourteen  days.  This 
curve  depends  upon  two  factors,  (1)  the  size  of  the 
dose  and  (2)  the  vital  resistance  of  the  patient.  A 
small  dose  may  have  to  be  repeated  frequently,  possi- 
bly every  day,  owing  to  the  short  duration  of  the 
"  positive  phase,"  whereas  a  large  dose  may  necessi- 
tate the  suspension  of  any  further  inoculations  on  ac- 


238 


APPLIED  IMMUNOLOGY 


count  of  a  prolonged  "  negative  phase,"  or  possibly 
the  establislmient  of  immunity  and  recovery.    Failure 


OPSONIC 
INDICL5 

HOUR                                 1 

11 

ll.is                   12 

12.22 

L3 

= 

1 

GOIVOCOCCUS 
TUBEJ^CLE    BACILLUS 

12 

< 

U 

0 
u 
( 

/ 

I 

NORMAL 

/ 

( 

.9 

/ 

.8 

/ 

.7 

/ 

/-. 

---^^, 

**/^ 

.6 

/ 

vj 

Fig.  33. — Case  of  gonorrhceal  arthritis  of  knee,  sho-wing  manner  of  differentiation 
between  the  possible  causes  of  synovitis  or  arthritis.  The  opsonic  indices  for  the  two 
suspected  etiological  organisms  were  determined  before  the  application  of  passive  hy- 
peraemia  to  the  infected  joint.  After  intermittent  application  for  one  and  a  half  hours 
the  indices  were  again  taken,  with  the  result  that  the  opsonic  index  for  the  gonococcus, 
the  specific  organism  in  this  case,  had  risen  considerably. 

to  evoke  a  rise  in  the  opsonic  index  may  be  attribut- 
able to  (1)  too  small  dosage,  (2)  reinoculations  dur- 
ing the  negative  phase,  or  (3)  inoculation  treatment 


THE  OPSONIC  INDEX 


239 


of  patients,  wherein  bacterins  are  contra-indicated, 

namely,  those  already  overwhelmed  by  their  infection. 

In  addition  to  its  value  as  a  control  of  treatment  in 

bacterin  therapy,  the  opsonic  index,  in  expert  hands. 


I 

T 

5    6    7    8    9   10  11  12  13  14  15  16  17  18  19  20 

2    3    4    5    6| 

2-7 
2-6 
25 
2-4 
2-3 
2-2 
2-1 
20 
V9 
18 
1-7 
1-6 
1-5 
t-4 
1-3 
12 
It 
10 

•9 
•8 
•7 
•6 
•5 
•4 

:3. 

40' 

38° 

3/ 
C 

36^ 

— 1 

e" 

^ 

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• 

1 

1 

• 

'  % 

1 

1 

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' 

' 

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A 

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\l 

'\ 

'^ 

1 

V 

« 

', 

\ 

I 

\ 

t 

j 

\ 

i 

1 

1 

\ 

1 

\ 

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f\ 

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^ 

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V 

f 

^ 

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\ 

/ 

/ 

^ 

^ . 

/ 

f 

\ 

"> — 

J 

/ 

/\ 

W 

■■ii" 

— 

\ 

^ 

/- 

A.  B. 

Fig.  34. — Pneumonia.     A,  recovery;     B,  death;     ,  curve  of  opsonins  (I); 

1 ,  curve  of  temperature  (T).     Observe  in  the  favorable  case  a  rise  in  the  opsonic 

index  coincidently  with  the  fall  in  temperature.     (Gigiioli  and  Stradotti.) 

has  been  utilized  both  diagnostically  and  prognosti- 
cally  ( Figs.  33  and  34 ) .  An  important  sphere  of  its 
utility  may  be  in  the  determination  of  the  degree  of 
animal  immunization  and  standardization  of  sera,  and 
has  been  so  employed  by  the  authors.    Its  value  in  a 


240 


APPLIED  IMMUNOLOGY 


diagnostic  and  prognostic  capacity  would  appear  to 
be  limited,  ( 1 )  because  other  methods,  as  the  cutane- 
ous allergic  reactions,  are  simpler  if  not  more  reliable, 
and  (2)  owing  to  a  certain  amount  of  error,  even  with 


FiQ.  35. — Showing  effect  of  variable    concentrations    of  bacterial  suspension  on 

determinations   of   opsonic   index.     .    bacterial   suspension    centrifuged   for    10 

minutes,  then  dilution  of  supernatant  suspension  so  that  1  c.mm.  contained  approxi- 
mately 10,000,000  bacteria.     ,  bacterial  suspension  centrifuged  for  10  minutes. 

.bacterial  suspension  centrifuged  for  10  minutes  and  supernatant  suspension 

diluted  with  3  volumes  of  0.85  per  cent,  saline  solution.  ,  bacterial  suspension 

standardized  by  McFarland's  "nephelometer." 

experienced  workers,  small  variations  may  not  be  de- 
pendable, although  it  renders  great  service  at  times,  in 
cases  of  mixed  infection,  enabling  the  immunologist 
to  choose  for  inoculation  the  particular  responsible 


THE  OPSONIC  INDEX  241 

bacterium,  or  in  the  course  of  immunization  to  select 
from  time  to  time  the  bacterin  to  which  the  index  is 
low.  It  is  generally  admitted  that  the  error  due  to 
technic  alone,  aside  from  the  question  of  personal 
equation,  amounts  to  one-  to  two-tenths.  This  varia- 
bility is  dependent  particularly  upon  ( 1 )  the  number 
of  cells  counted  and  ( 2 )  the  concentration  of  the  bac- 
terial suspension  (Fig.  35). 


16 


XXI 

PRACTICAL  APPLICATION  OF  BACTERIAL  INOCU- 
LATION IN  MEDICINE,  PROPHYLACTICALLY 
AND  THERAPEUTICALLY 

GENERAL  CONSIDERATIONS— INDUCED  AUTO-INOCULA- 
TION—DURATION OF  ACTIVE  IMMUNITY"— MODES  AND 
TECHNIC  OF  ADMINISTRATION  OF  BACTERINS— 
DOSAGE— CONTRA-INDICATIONS,  LIMITATIONS  AND 
CAUSES  OF  FAILURE  IN  BACTERIN  THERAPY— APPLI- 
CATION AND  RESULTS  OF  BACTERIAL  INOCULATIONS 
IN  SPECIAL  DISEASES— DISEASES  OF  THE  SKIN  AND 
SOFT  PARTS— DISEASES  OF  THE  GENITO-URINARY 
SYSTEM— DISEASES  OF  BONES  AND  JOINTS— DISEASES 
OF  THE  EYE,  EAR,  NOSE  AND  THROAT— DISEASES  OF 
THE  LUNGS— DISEASES  OF  THE  ALIMENTARY  SYSTEM- 
DISEASES  OF  THE  CARDIOVASCULAR,  LYMPHATIC  AND 
NERVOUS  SYSTEMS,  ALSO  OTHER  ACUTE  SPECIFIC 
FEVERS— MALIGNANT  NEOPLASMATA— YEAST  AND 
SOUR  MILK 

General  Indications. — Wright  has  declared  that, 
"  we  have  in  the  power  of  increasing  the  antibacterial 
power  of  the  blood  by  the  agency  of  vaccines,  and  in 
our  power  of  bringing  the  antibacterial  agencies  of 
the  blood  into  operation  in  the  focus  of  infection,  be- 
yond all  comparison  the  most  valuable  assets  of  medi- 
cines." It  might  be  wise  to  modify  this  assertion  to 
the  effect  that,  in  the  competent  employment  of  bac- 
terins  tve  have  one  of  the  most  valuable  assets,  A 
very  important  fact  ever  to  be  remembered  in  the 
practice  of  medicine — and  this  includes  particularly 
bacterin  therapy — is  never  to  discard  or  disregard  old- 
established  measures  of  proven  worth,  for  a  new  idea, 

242 


BACTERIAL  INOCULATION  243 

no  matter  how  attractive  and  possibly  of  superior 
value,  for,  by  the  skilful  association  of  all,  cure  will 
be  best  promoted  and  facilitated.  Wright's  doctrine 
has  become  classical:  "  The  medical  man  who  has  re- 
course to  vaccine  therapy  ought  to  have  familiar  ac- 
quaintance with  the  microbes  which  affect  the  human 
body.  He  ought  to  appreciate  the  fact  that  vaccines 
owe  their  efficacy  to  the  reaction  they  set  up  in  the 
tissues,  and  not  to  any  action  exerted  directly  by  the 
vaccine  upon  the  invading  microbe.  He  ought  to  have 
mastered  the  general  principles  of  immunization.  He 
ought  to  know  in  connection  with  each  vaccine  the 
minimum  effective  dose,  i.e.,  the  dose  which  gives  the 
minimum  immunizing  reaction  without  any  interven- 
ing negative  phase ;  and  the  medium  or  average  dose, 
i.e.,  the  dose  that  gives,  after  a  negative  phase,  a  more 
powerful  immunizing  reaction.  He  ought  to  know  the 
general  conditions  which  affect  the  sensibility  of  the 
organism.  He  ought  to  understand  how  to  adjust  the 
dose  to  the  requirements  of  the  individual  patient, 
and  he  ought  to  have  a  knowledge  of  the  conditions 
which  obtain  in  the  focus  of  infection,  and  of  the 
methods  of  circumventing  the  difficulties  which  are 
introduced  by  these  conditions." 

It  is  extremely  improbable  that  bacterin  therapy 
as  practiced  by  the  average  general  practitioner  is 
destined  to  realize  the  full  measure  of  its  promise. 


244  APPLIED  IMMUNOLOGY 

either  because  he  lacks  the  required  bacteriological 
facilities  or  is  too  busy  to  devote  the  necessary  time 
and  attention  to  trivial  signs  and  symptoms.  It  seems 
not  to  be  appreciated  by  physicians  generally  that  bac- 
terins  are  agents  capable  equally  for  good  or  evil.  It 
cannot  be  too  strongly  emphasized  that  bacterin 
therapy  is  merely  a  valuable  accessory  to  Nature  in 
the  art  and  science  of  healing  and  is  not  a  "  cure  all." 
Carelessness,  ignorance,  incompetence  or  a  desire  on 
the  part  of  the  therapeutist  to  push  the  treatment, 
when  beneficial  results  become  apparent,  will  lead  not 
only  to  failure,  perhaps  disaster,  but  at  all  times  dis- 
credits a  therapeutic  measure  of  great  value,  and  de- 
prives the  patient  of  his  natural  resources  for  re- 
covery. Two  solutions  for  the  problem  are  offered 
the  general  practitioner  contemplating  bacterial  in- 
oculations :  either  to  familiarize  himself  more  with  im- 
munology, including  bacteriology  and  laboratory 
methods,  or  to  refer  his  patient  to  or  cooperate  with 
an  immunologist,  precisely  as  he  is  accustomed  at  times 
to  consult  an  ophthalmologist. 

Induced  Auto-inoculation, — Therapeutic  inocula- 
tion may  assume  one  of  two  forms,  (1)  exogenous 
injection  of  bacterial  suspensions,  forming  the  main 
theme  of  this  chapter,  and  ( 2 )  endogenous  inoculation 
or  auto-inoculation.  The  latter  is  a  very  common  clini- 
cal phenomenon  and  may  be  spontaneous  or  induced. 


BACTERIAL  INOCULATION 


245 


Spontaneous  auto-inoculation  is  frequently  observed 
in  a  patient  the  victim  of  subacute  or  chronic  phthisis 
pulmonalis,  and  is  characterized  by  the  irregular  flares 
of  temperature.  If  the  doses  of  bacteria  or  their  prod- 
ucts, which  at  these  times  are  cast  off  into  the  general 
circulation,  are  too  great  or  repeated  too  often,  the  pa- 


11 


i 


5 


I 


s 


16- 


I 


si 


11 


^ 


Fig.  36. — Case  of  gonococcal  arthritis.  Showing  the  effect  of  massage  on  the  gono- 
cocco-opsonic  index.  I  =inoculation  of  30,000,000  gonococci.  M  =massage.  (Dr. 
J.  Freeman.) 

tient  is  thrust  into  a  profound  or  permanent  "  nega- 
tive phase  "  and  death  supervenes.  If,  on  the  other 
hand,  the  dose  of  re-infection  be  of  the  proper  size 
and  occurs  opportunely,  the  patient  becomes  to  a  cer- 
tain extent  immunized  and  at  least  a  temporary  re- 
covery takes  place.  Induced  or  artificial  auto-inocula- 
tion is  a  most  interesting  problem  and  at  times  has 


246 


APPLIED  IMMUNOLOGY 


proved  feasible  in  the  treatment  of  various  infections. 
It  would  appear  advisable  ofttimes  when  bacterin  or 
vaccine  therapy  is  contra-indicated.  It  consists  in  an 
endeavor  to  inoculate  the  patient  from  his  own  lesion 
by  purely  physical  measures.  These  include  massage, 
exercise,  applications  of  heat,  passive  hyperaemia,  etc.. 


TUBERCULO- 
OPSONIC 


inULA. 

~ 

■       "" 

- 

""■ 

'~~ 

! 

iiu 

< 

>• 

CO 

1 

< 

IS 

"5^ 

14 

/ 

!-3 

/ 

\ 

\ 

< 
<0 

12 

-J 

\ 

/ 

c*^ 

\ 

L 

II 

? 

1 

\ 

/ 

5 

k 

5 

Y 

NORMAL  10 

/ 

\ 

'Jr_ 

\ 

— 

•9 

1 

\ 

A 

*V 

N, 

\ 

8 

/ 

V 

\ 

i 

> 

h  ^- 

•7 

•6 

FEB. 

21 

22 

23 

24- 

25 

26    27 

28 

1 

2 

3 

Fia.  37. — Case  of  tuberculous  cervical  lymphadenitis.     Showing  effects  of  massage 
on  tuberculo-opsonic  indices.     (Wright.) 

and  explains  wherein  lies  the  diagnostic  value  of  the 
opsonic  index,  for  example,  in  gonorrhoeal  arthritis 
(Figs.  33  and  36) ,  and  in  tuberculous  cervical  lymph- 
adenitis (Fig.  37) .  The  production  of  induced  auto- 
inoculations  may  result  in  excessive  dosage,  as  we 
have  no  means  to  gauge,  at  the  moment,  the  size  of 


BACTERIAL  INOCULATION  247 

the  dose  cast  into  the  circulation,  and  in  view  of  the 
fact  that  the  inoculation  consists  of  living  bacteria, 
not  as  in  ordinary  bacterin  therapy  of  measured  dead 
microorganisms,  the  method  is  attended  by  a  certain 
amount  of  danger  and  should  be  utilized  only  by  ex- 
perienced hands. 

Duration  of  Active  Immunity, — In  general  it  is 
admitted,  and  may  be  stated,  that  the  greatest  and 
most  lasting  immunity  is  produced  by  inoculations  with 
living  bacteria,  then  with  dead  bacteria  and  finally  with 
the  products  of  bacterial  growth.  This  has  been  quite 
satisfactorily  demonstrated  by  animal  experimenta- 
tion, and  a  common  procedure  is  to  begin  the  process 
of  immunization  with  inoculations  of  dead  bacteria, 
followed  by  the  living  microorganisms.  Thus  far, 
the  profession  has  not  seen  fit,  nor  would  patients  sub- 
mit to  injections  of  living  bacteria,  although  by  re- 
sort to  the  technic  of  Williams  and  Webb — inocula- 
tions at  first  with  a  single  living  bacterium,  then  by 
cautious  count  progressively  increasing  the  dosage — ■ 
or  supplementing  inoculations  of  dead  by  living  or  at- 
tenuated bacteria,  no  danger  should  be  incurred,  and 
the  day  may  not  be  far  remote  when  such  a  practice 
in  expert  hands  will  be  an  unobjectionable  and  com- 
mon procedure. 

Immunity  is  never  absolute,  but  the  protection  af- 
forded by  bacterial  inoculations  lasts  indefinitely,  fre- 


248  APPLIED  IMMUNOLOGY 

quently  for  months  or  years.  There  is  substantial  evi- 
dence that  prophylactic  typhoid  immunization  pro- 
tects the  individual  for  at  least  three  years.  The 
duration  of  immunity  may  be  determined  by  cutane- 
ous allergic  tests  and  by  serological  reactions,  includ- 
ing the  opsonic  index. 

Modes  and  Technic  of  Administration  of  Bac- 
terins. — Bacterins  have  been  administered  subcutane- 
ously,  intravenously,  orally  and  per  rectum.  The 
last  three  methods  are  not  in  general  use,  in  spite  of 
sporadic  marvellous  results,  nor  do  they  appeal 
strongly  on  immunological  grounds,  and  consequently 
seem  not  destined  to  become  popular.  Subcutaneous 
administration  is  the  procedure  usually  practiced.  In 
the  event  of  mixed  infections,  multiple  bacterins  of 
the  respective  pure  bacterial  cultures  may  be  prepared 
for  alternate  or  indicated  inoculations,  or  a  mixed 
bacterin  proportionately  compiled  of  the  various  in- 
vading microorganisms  may  be  employed.  The  technic 
consists  in  sterilizing  the  skin  with  a  pledget  of  cotton 
and  alcohol  overlying  the  insertion  of  the  deltoid 
muscle  or  elsewhere  on  the  body,  as  described  on  page 
178.  It  is  recommended  whenever  possible  to  inoc- 
ulate on  the  distal  side  of  the  lesion,  thereby  giving  the 
patient  the  immediate  benefit  of  the  antibody  forma- 
tion through  the  local  lymph  system  in  addition  to 
the  subsequent  effect  to  be  obtained  through  the  blood- 


BACTERIAL  INOCULATION  249 

serum.  The  syringe  (Fig.  38)  constructed  entirely  of 
glass  or  one  having  an  asbestos  piston  is  most  service- 
able, as  it  permits  of  ready  cleansing  and  sterilization 
by  boiling.  When  inoculations  are  given  on  a  large 
scale,  requiring  frequent  sterilization,  the  oil  method 
of  Wright  is  novel,  convenient  and  effective.  It  con- 
sists in  heating  a  quantity  of  olive  oil  over  a  gas  flame. 
The  oil  will  heat  to  150°  C.  without  boiling.  This  is 
one  and  a  half  times  the  temperature  of  boiling  water 
and  instantly  sterilizes  the  syringe  when  drawn  up  and 
forced  out  after  inoculation.    From  the  ampoule  after 


Fig.  38. — All-glass  hypodermic  syringe.     Desirable  for  bacterial  inoculations  because 
of  ease  of  cleansing  and  surety  of  sterilization. 

filing  or  breaking  off  its  neck  or  from  the  small  bottles, 
• — in  the  case  of  the  rubber-capped  one  by  plunging 
the  needle  through  a  drop  or  two  of  phenol,  formalin 
or  alcohol  placed  on  the  rubber, — the  desired  dose  of 
bacterin  is  drawn  up  into  the  syringe.  In  the  case  of 
some  vials  with  rubber  stoppers  it  is  expedient  to  with- 
draw partially  the  piston  of  the  syringe  before  thrust- 
ing it  through  the  cork,  then  to  push  it  home  and  as  it 
is  slowly  withdrawn  again  the  desired  quantity  of  bac- 
terin readily  follows  the  piston.  If  the  skin  is  firmly 
grasped  and  lifted  between  the  finger  and  thumb  and 


250  APPLIED  IMMUNOLOGY 

a  perfectly  sharp  pointed  fine  needle  is  employed,  the 
amount  of  pain  occasioned  by  the  puncture  is  ex- 
tremely slight.  The  bacterin,  if  a  large  dose,  should 
be  slowly  injected  into  the  loose  subcutaneous  tissue 
to  minimize  pain.  As  the  needle  is  withdrawn  the 
site  of  the  pmictui-e  should  be  compressed  and  the 
skin  gently  stroked  a  couple  of  times  with  the  pledget 
of  alcoholized  cotton.  No  further  dressing  is  neces- 
sary or  desirable. 

Dosage. — The  size  of  the  dose  of  bacterin  must  be 
cautiously  gauged.  The  two  fundamental  considera- 
tions are  ( 1 )  the  nature  of  the  particular  infecting  bac- 
terium and  (2)  the  condition  or  state  of  the  patient. 
It  should  be  thoroughly  realized  that  not  only  bacteria 
in  general  but  individual  strains  of  the  same  bacterium 
differ  greatly  in  virulence  and,  as  a  rule,  the  more  viru- 
lent the  microbe  the  smaller  should  be  the  inocula- 
tion. To  determine  this  and  the  inmiunizing  power  of 
the  bacterin,  with  the  greatest  precision,  various  clini- 
cal laboratory  and  blood  tests  are  essential.  Thus  the 
dose  of  the  more  virulent  bacteria,  as  the  streptococcus, 
pneumococcus,  gonococcus,  colon  bacillus,  etc.,  should 
be  considerably  smaller,  that  is,  one-half  to  one-fom'th 
that  of  the  less  virulent  microorganisms,  as  the  staphy- 
lococcus. Again,  a  most  important  problem  is  the  de- 
termination of  the  viiimnal  effective  and  the  average 
and  maocimal  dose  for  each  bacterium.     In  desper- 


BACTERL4L  INOCULATION 


251 


ately  or  acutely  ill  patients,  or  with  patients  in  whom 
a  beneficial  response  has  been  obtained  with  the  mini- 
mal effective  dose,  this  should  not  be  exceeded.  With 
chronic  cases,  presenting  well-localized  lesions,  the 
average  or  maximal  dose  may  be  administered  at  the 
start,  and  subsequently  increased.  The  following 
table,  giving  the  minimal,  average  and  maximal  doses 
employed  in  the  authors'  experience,  may  prove  of 
service : 

THERAPEUTIC  DOSE  TABLE 


Bacterin 


Gonococcus 

Colon 

M.  Catarrhalis 

Influenza 

Typhoid 

Pneumococcus 

Streptococcus 

Staphylococcus 

B.  Punctatum 

B.  Proteus  Vulgaris. . . 

B.  Friedlander 

B.  Acnes 

Pertussis 

Diphtheria 

Pseudodiphtheria  .... 

B.  Pyocyaneus 

B.  Fluorescens 

B.  Pseudotuberculosis 

Rodentium 

B.  Acidi  Lactici 

B.  Lactis  Aerogenes. . . 

B.  Mallei... 

B.  Fusiformis 

B.  Koch- Weeks 

B.  Morax-Axenfeld .  .  .  , 
Tuberculin 


Actinomycotin . 


Minimal  effective  dose 


Average  dose 


2  to 
5  to 

10  to 
5  to 
5  to 

10  to 
2  to 

50  to 

10  to 
5  to 

10  to 
5  to 
5  to 
5  to 

10  to 


5  million 
10  million 
25  million 
10  million 
10  million 
20  million 
5  milHon 
100  million 
25  million 
10  million 
20  million 
10  million 
10  million 
10  million 
25  million 


25  million 


2  to 
10  to 


5  million 
25  million 


0.00001  to  0.0001 

milligram 
0.001  milligram 


50  to 
25  to 
50  to 
25  to 
25  to 
25  to 
25  to 
100  to 
100  to 
50  to 
50  to 
25  to 
25  to 
25  to 
50  to 
50  to 
50  to 


100  million 

50  million 

100  million 

50  million 

50  million 

50  million 

50  million 

400  million 

200  million 

100  million 

75  million 

100  million 

100  million 

50  million 

100  million 

100  million 

100  million 


Maximum  dose 


50  to  100  million 
50  to  100  million 
50  to  100  million 

50  to  100  million 
50  to  100  million 
50  to  100  million 
0.0001  to  0.001 

milligram 
0.01  to  0.1  milli- 
gram 


400  million 
400  million 
300  million 
200  million 
500  million 
300  milHon 
200  million 
1000  million 
400  million 
400  million 
300  million 
200  million 
200  million 
100  million 
300  million 
400  million 
400  million 

400  million 
400  million 
400  million 

400  million 

400  million 

250  million 

100  milligrams 

4  milligrams 


252  APPLIED  IMMUNOLOGY 

The  physical  and  clinical  state  of  the  patient, 
apart  from  the  variability  in  the  virulence  of  bacteria, 
ofttimes  presents  a  difficult  problem  as  to  the  correct 
dosage  of  bacterin.  In  this  connection,  the  age,  sex, 
and  stature  of  the  patient,  the  gravity  of  his  illness, 
the  acuity  or  chronicity  of  the  affection,  a  non-febrile 
or  febrile  condition  and  his  debilitated  or  toxic  state 
demand  the  keenest  discrimination.  The  golden  rule 
is  the  sicker  the  patient  the  smaller  the  dose,  but  both 
the  clinical  symptomatology  and  the  opsonic  index  may 
suffice  to  determine  that  he  is  too  sick  for  any  dose, 
however  infinitesimal. 

Subsequent  inoculations  both  as  to  size  of  doses  and 
intervals  may  be  governed  in  most  diseases  by  close 
and  accurate  observation  of  the  clinical  symptoms  or 
by  the  revelations  of  opsonic  indices  or  by  the  utiliza- 
tion of  both  of  these  controls,  an  advisable  procedure 
for  the  best  results  in  no  small  number  of  cases. 
Generally  speaking,  the  clinical  symptomatology  is 
of  primary  importance  and  the  opsonic  index  takes 
second  place ;  at  times,  however,  these  become  reversed. 
It  is  an  easy  matter  to  control  properly  the  process 
of  immunization  by  study  of  the  clinical  symptoms, 
and  success  will  obtain  only  by  an  experienced  and 
careful  observation  of  such  trivial  phenomena  as 
malaise,  indisposition  to  work  or  play,  bodily  aches, 
grippe-like  attacks,  headache,  anorexia,  slight  fever. 


BACTERIAL  INOCULATION  253 

nausea,  variations  in  weight,  leucocytic  and  hsemo- 
globin  determinations,  and  particularly  local  manifes- 
tations in  the  diseased  region  as  well  as  at  the  sites  of 
inoculations.  Allusion  has  previously  been  made  to  the 
fact  that  there  are  many  diseases  in  which  guidance 
furnished  by  determinations  of  the  opsonic  index  is 
indispensable  for  the  proper  execution  of  bacterin 
therapy,  and  without  which  bacterial  inoculations,  per- 
haps, had  best  not  be  practiced.  These  include  espe- 
cially deep-seated  affections,  as  bronchitis,  pneumonia, 
certain  intestinal  infections  and  diseases  of  the  genito- 
urinary tract  typified  by  pyelitis,  cystitis,  seminal 
vesiculitis,  prostatitis,  etc.  The  safest  rule  to  be  ob- 
served respecting  bacterin  therapy,  controlled  only 
by  the  clinical  symptomatology,  is  to  begin  with  a 
very  small  or  assuredly  harmless  dose;  if  there  be 
absolutely  no  reaction,  local,  focal  or  general,  in  two 
or  three  days  a  second  inoculation,  twice  the  size  of 
the  first,  may  be  given,  and  so  on  with  intervals  of 
three  to  seven  days  until  reactionary  phenomena  are 
observed.  The  slightest  reaction  is  evidence  that  the 
dose  has  been  sufficient  if  not  too  large  and  indicates 
that  no  further  inoculations  are  to  be  made  until  all 
reactionary  signs  have  completely  subsided  for  several 
days.  Frequently,  the  next  inoculation  must  be  de- 
ferred for  two  or  more  weeks  and  when  administered 
should  not  exceed  in  size  its  predecessor,  commonly 


254  APPLIED  IMMUNOLOGY 

being  cut  down  to  only  one-half  of  the  previous  dose. 
Strict  observance  of  this  dictum  is  imperative,  but 
unfortunately  not  in  accordance  with  the  instructions 
furnished  the  general  practitioner  by  all  pharmaceuti- 
cal firms  marketing  stock  bacterins,  thus  accounting 
for  no  small  nmiiber  of  failures  in  bacterin  therapy. 
No  hard  and  fast  rule  can  be  dogmatically  laid  down, 
either  as  to  interval  or  size  of  the  inoculations.  Each 
case  is  a  study  unto  itself  and  must  be  treated  ac- 
cordingly. 

Contra-indications,  Limitations  and  Causes  of 
Failure  of  Bacterin  Therapy, — By  virtue  of  the 
theory  of  biological  therapeusis,  little  should  be  ex- 
pected of  bacterial  inoculations  in  the  acute  stages  of 
infectious  diseases,  indeed  they  may  exert  an  evil  in- 
fluence. Bacterin  therapy,  however,  is  particularly 
contra-indicated  when  the  individual  is  overwhelmed 
by  a  diffuse  infection  or  when  owing  to  prolonged 
illness  he  is  prostrated  or  his  tissues  are  greatly  wasted 
and  no  longer  susceptible  to  artificial  stimulation  for 
the  production  of  antibodies.  Such  states  are  bacte- 
rsemia,  septicaemia,  pysemia  and  saprsemia.  Among 
other  contra-indications  should  be  mentioned  igno- 
rance and  inexperience  on  the  part  of  the  would-be 
immunologist,  and  complicating  surgical  conditions 
demanding  primary  and  immediate  operative  inter- 
vention. 


BACTERUL  INOCULATION  255 

Bacterin  therapy  has  definite  and,  in  the  minds  of 
immunologists,  well-recognized  limitations.  It  is  not 
assumed  to  be  a  specific,  capable  of  cm'e  in  all  cases 
to  the  exclusion  of  other  important  time-honored 
therapeutic  measures,  physical  and  chemical.  It  pur- 
ports to  be  merely  the  logical  and  scientific  means  to 
assist  Natm-e  in  her  struggle  against  infection,  and, 
with  due  appreciation  and  application  in  this  light,  it 
will  seldom  fail  to  render  due  service.  It  offers  no 
promise  to  resuscitate  a  medical  derelict. 

'^4^11usion  must  be  made  to  certain  causes  of  failure 
by  those  practicing  bacterial  inoculations.  Sum- 
marily, they  may  be  stated  to  be:  (1)  utilization  of  the 
improper  bacterium,  whether  autogenous  or  hetero- 
geneous; (2)  routine  employment  of  stock  instead  of 
autogenous  bacterins;  (3)  ignorance  in  administra- 
tion, either  of  size  of  doses  or  intervals  of  inoculations, 
and  (4)  disregard  of  commonly  associated  conditions. 
The  mistake  of  utilizing  the  wrong  germ  could  be 
obviated  by  the  routine  employment  of  autogenous 
bacterins  whenever  possible.  This  implies  technical 
laboratory  and  bacteriological  knowledge  by  each 
physician  contemplating  bacterin  therapy  or  that  he 
consult  with  or  refer  his  patient  to  an  immunologist. 
Thus  there  would  arise  no  shifting  of  responsibility 
between  the  laboratory  worker  making  and  stand- 
ardizing the  bacterial  suspension  and  the  general  prac- 


^56  APPLIED  IMMUNOLOGY 

titioner  desiring  to  give  the  inoculations.  Otherwise, 
in  the  event  of  an  unhappy  result,  the  former  will 
accuse  the  latter  of  incompetence  and  the  latter  the 
former  of  faulty  technic.  This  seems  like  stringent 
doctrine,  but  we  believe  in  the  long  run  will  redound 
not  only  to  the  credit  of  physicians  and  bacterin 
therapy,  but  also  and  especially  to  the  better  health 
of  patients.  Even  in  the  experience  of  expert  bac- 
teriologists it  becomes  at  times  no  easy  task  to  isolate 
from  a  mixed  infection  the  fundamental  etiological 
bacterium.  Obviously,  a  bacterin  prepared  from  a 
superimposed  or  secondary  infection,  even  though  that 
bacterium  be  preponderant,  would  produce  little  or  no 
effect,  curatively,  upon  the  primary  or  underlying 
morbid  process.  Stock  bacterins,  if  employed — a  pro- 
cedure at  times  advisable — should  be  polyvalent,  that 
is,  constituted  by  as  many  strains  as  may  be  obtain- 
able of  the  particular  species  of  bacterium  in  question. 
Caution  should  always  be  exercised  to  determine  by 
bacteriological  methods  the  exact  identity  of  the  in- 
fecting bacterium  in  a  given  case  before  proceeding 
with  the  ''  supposedly  correct  "  stock  bacterin.  On 
more  than  one  occasion  we  have  found  patients  falsely 
immunized  with  a  stock  preparation  devoid  of  the 
bacterium  actually  causing  the  disease.  Such  failures 
discredit  physicians  vastly  more  than  the  mode  of 
therapy.    The  advantage  of  the  autogenous  prepara- 


BACTERIAL  INOCULATION  257 

tion  is,  naturally,  that  the  patient  will  be  immunized 
with  the  exact  strain  of  the  germ  by  which  he  has  been 
infected,  leading  to  a  more  definite  and  decisive  re- 
sult. The  commercial  so-called  ''  mixed  bacterins  or 
vaccines "  for  therapeutic  purposes  cannot  be  too 
highly  condemned,  simply  on  scientific  grounds; 
prophylactically  their  employment  would  appear 
rational.  It  is  not  true  that  the  injection  of  a  few 
million  extra  brands  of  germs,  in  addition  to  that 
responsible  for  the  main  affection  or  possibly  con- 
comitant infections,  is  without  deleterious  effect. 
Each  variety  of  bacteria  thus  injected  stimulates  the 
tissue  cells  to  the  production  of  its  specific  antibody. 
This,  certainly,  is  an  unnecessary  work  or  drain  on 
the  part  of  the  immunological  mechanism,  so  far  as 
the  irrelevant  bacteria  are  concerned,  and  has  no  ef- 
fect therapeutically  upon  the  pathological  process. 
Again,  the  bacterin,  especially  if  heterogeneous,  may 
be  unfit  for  use,  that  is,  too  old  or  in  a  state  of  precipi- 
tation or  autolysis,  or  chemically  contaminated  from 
its  container,  be  it  bottle,  vial,  ampoule  or  syringe. 

Finally  an  item  of  much  importance  in  the  prep- 
aration of  a  bacterin,  whether  autogenous  or  stock, 
is  that  it  should  not  be  overheated,  thereby  destroy- 
ing its  immunizing  properties.  The  correct  degree  of 
temperature  is  merely  the  thermic  death  point  for  the 
respective  bacterium. 

17 


258  APPLIED  IMMUNOLOGY 

Many  failures  in  therapeutic  inoculation  result 
from  improper  administration  of  the  bacterin.  The 
doses  given  are  either  too  large  or  too  small,  more 
frequently  the  former,  and  the  intervals  carelessly 
spaced.  This  is  partially  accounted  for  by  the  fact 
that  some  physicians  are  gifted  with  superior  judg- 
ment and  possess  keen  powers  of  observation,  while 
others  are  below  par,  yet  overworked  and  indifferent, 
or  overenthusiastic  or  prejudiced.  Another  impor- 
tant cause  of  failure  in  biological  therapeusis  is  dis- 
regard of  associated  conditions.  These  are  both 
general  and  local.  The  general  comprise  diabetes,  in- 
testinal stasis,  constipation  and  toxicosis.  In  these 
states  phagocytosis  is  markedly  handicapped  or  im- 
possible. The  local  causes  responsible  for  failure 
include  marked  suppurative  foci,  caries,  sequestra  and 
other  conditions  demanding  primary  surgical  interven- 
tion. Again,  owing  to  the  anatomy  of  the  part,  as  in 
chronic  otitis  media,  also  on  account  of  the  pathology 
as  observed  in  sinuses,  fistula  and  old  abscess  cavities, 
where,  due  to  coagulated  fibrin  and  old  inflammatory 
exudate  and  cicatrization,  the  diseased  area  becomes 
walled  off,  it  is  impossible  for  the  systemic  blood- 
serum,  even  though  highly  opsonized,  to  bathe  the 
affected  part,  hence  exert  a  healing  influence.  Here 
the  rational  employment  of  hyperemia,  by  massage, 
rubefacients,  heat  and  Bier's  methods,  flushing  sinuses, 


BACTERIAL  INOCULATION  259 

abscesses,  etc.,  with  Wright's  solution  (one  and  a  half 
per  cent,  sodium  citrate  in  five  per  cent,  saline),  in- 
cision, citric  acid  to  decrease  the  viscosity  of  the  blood, 
nuclein  and  eliminants  and  tonics  directed  to  the  in- 
testinal tract,  kidneys,  skin  and  liver,  become  indis- 
pensable adjuvants,  converting  an  otherwise  sure  fail- 
ure into  natural  victory. 

Application  and  Results  of  Bacterial  Inoculations 
in  Special  Diseases. — During  the  past  decade,  owing 
to  the  opsonic  tidal  wave  and  the  resultant  interest  in 
bacterial  inoculation,  injections  of  killed  bacteria  have 
been  made,  both  in  a  protective  and  curative  capacity, 
in  almost  all  infections  or  infectious  diseases  to  which 
man  falls  heir.  There  is  not  a  system  in  the  human 
organism  to  which  bacterin  therapy  has  not  been  found 
applicable.  Just  as  was  the  case  with  tuberculin 
therapy  in  the  early  nineties,  the  therapeutic  pendu- 
lum swung  too  far,  but  to-day  the  cordon  is  being 
drawn  tighter  and  tighter  around  those  specific  dis- 
eases amenable  to  this  form  of  therapy  and  year  by 
year  the  indications  and  contra-indications  and  modus 
operandi  become  better  known  and  the  doctrine  of  bac- 
terial inoculation  more  intelligently  engrafted. 

Diseases  of  the  Skin  and  Soft  Parts 
This  group,  comprising  acne,  boils,  carbuncles, 
abscesses,  ulcers,  cellulitis,  dermatitis,  impetigo,  syco- 
sis, sinus,  fistula,  tuberculosis,  glanders,  actinomycosis, 


260 


APPLIED  IMMUNOLOGY 


smallpox  and  bubonic  plague,  by  all  odds  furnishes 
the  most  fertile  field  for  bacterial  inoculations,  thera- 
peutically and  prophylactically. 

Acne. — Extraordinary  results  have  followed  bac- 
terin  therapy  in  this  disease,  especially  in  the  pustular 
variety,  when  usually  the  M.  alhus,  but  occasionally 
the  M,  aureus  and  rarely  the  M,  citreus,  have  been 
isolated  (Fig.  39).     Incipient  or  non-pustular  acne, 


tNDEX 

OCT        NOV       H^          U^          NOV            0|C               DEC                         JAN. 

™ 

?..?. 

?> 

®«  OPSONIC    INDEX                                                                                                   ,              /•  ■ 

2.0- 

X"  INOCULATION    M.ALBUS                                                                                                   / 

1  .1 

1  .8 

o                      O                       O                       OO                     OO                    ooo                   o 

-°- -5 S- S- § %—Z' S— S-S- o— 

2                     ri                    -n                     o                 o ._.o            ,-, _o o       o     .      ..^_     - 

o                   OO                    o               o                  OO                 o_oo                o^7 

-^ s( ^ j( s S— S ^^2— ci'-:f'^ ^f- 

I  .7 

1  .U 

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1  .4 

J. .a 

^y*^                                                                     "^ 

1  .1 

1=^^  \                                  / 

1  . 1 

/         \                                    / 

/          \                              ^ 

/                         \                                             JL-^ 

fi.R 

«r               \                  .*<>'' 

y                              V                  ^sr 

O.fc 

<*                                \  s^ 

QjT 

J 

Fig.  39. — Harris  E.  T.     Case  of  long-standing  and  obstinate  pustular  acne  vulgaris. 
Yielded  to  autogenous  bacterin  therapy  only  after  a  prolonged  course  of  treatment. 

according  to  the  researches  of  Unna  and  Sabouraud 
caused  by  the  Bacillus  acnes,  is  in  many  cases  favor- 
ably influenced  by  inoculations  with  this  organism,  al- 
though the  results  are  not  nearly  so  brilliant  as  with 
the  staphylococci  in  the  pustular  form  of  the  disease. 
Sabouraud  contends  that  the  acne  bacillus  is  also  the 
cause  of  sehorrhoea  and  alopecia  areata,  and  Fleming 
has  reported  signal  successes  in  the  treatment  of  these 
conditions  with  B.  acnes  bacterin.    In  the  treatment  of 


BACTERIAL  INOCULATION 


261 


acne  in  any  form,  the  accessory  or  supplemental 
measures  are  of  prime  consideration,  and  include  cor- 
rection of  underlying  systemic  conditions,  notably  ali- 
mentary and  genital  disorders,  lymphagogues  as 
sodium  citrate  or  citric  acid  internally  and  hot  fo- 
mentations, the  removal  of  comedones  and  pustules 
locally,  followed  by  sulphur,  salicylic  acid  or  betanaph- 
thol  ointment,  etc. 


NOEXM 

50  1  I  S  4  5  t 

IN 

JUUY 

:  i  6 10 11  uij  14 15  It  in 

H:0!.1U23M25it2Tyi9  50  51 

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Fig.  40. — Annie  H.  Furunculosis  of  nostril.  _  It  will  be  observed  here  that  the 
inoculations  were  governed  entirely  by  the  clinical  course  of  the  case;  the  opsonic 
indices  were  taken  incidentally,  merely,  to  note  when  the  patient  reached  the  "high 
tide  of  immunity, "  so  that  further  inoculations  might  be  suspended. 

Furunculosis  and  Carhunculosis. — None  the  less 
remarkable  are  the  effects  of  bacterial  inoculations  in 
these  common  affections.  The  infecting  bacterium  is 
almost  invariably  the  M,  aureus.  In  the  case  of  the 
acute  primary  boil  or  carbuncle,  bacterin  therapy  need 
not  and  should  not  be  used  except  by  an  expert  pos- 
sessing a  knowledge  of  the  opsonic  index,  since  more 
harm  than  good  may  result  (see  page  226),  whereas 


262 


APPLIED  IMMUNOLOGY 


in  the  chronic  recurrent  form  of  the  diseases  it  is  the 
most  valuable  therapeutic  agent  at  our  disposal.  Here 
again  opsonic  indices  utilized  to  guard  against  over- 
inoculation,  and  to  govern  remote  inoculations,  will 
suffice  to  prevent  recurrences  (Figs.  40  and  41) . 

Abscesses. — These  may  be  primary  or  complicate 
other  diseases,  as  typhoid  fever.  The  bacteria  isolated 
include  31.  aureus,  alhus  and  citreus.  Streptococcus 
pyogenes,  Pneumococcus  lanceolatus,  B.  coli,  B.  ty- 


INDEX 

1.3 

l.^ 

1.1 

1.0 

.9 

.8 
.7 
.6 

4 

^3 

s 

- 

1^ 

1 

i:MBE 

4'i 

— ; 

19 
Ik 

n 

u 

n 

M 

?Q 

1 

0 

4 

r 

% 

1 

s 

^ 

F 

^ 

-^ 

So 

g 

_J 

-^ 

' 

■' 

t1 

& 

r^^ 

^ 

©= 

©•OP.^ONin   INHFX 

y-lNOLSTAPHYLnrQCCl 

Fig.  41. — Fred.  G.  Carbuncle  of  neck.  Observe  in  this  case  the  utilization  of  the 
opsonic  index  to  determine  the  maintenance  of  immunity  in  the  "positive  phase," 
after  the  suspension  of  inoculations. 

phosus,  B,  pyocyaneus,  B,  tuberculosis,  B,  antkracis, 
B,  mallei  and  Streptothrioo  actinomyces.  An  autoge- 
nous bacterin  is  always  preferable  to  a  stock  prepara- 
tion and  the  therapy  when  so  conducted  usually  re- 
sults brilliantly  (Figs.  42  and  43).  Little  should  be 
expected  from  bacterial  inoculations  with  secondary 
invaders,  as  B.  pyocyaneus,  B,  lactis  aerogenes,  etc.,  or 
with  B.  anthracis,  B.  mallei  and  Streptothrix  actino- 
mycosis. 


FiQ.  43. — Linda  c'  ""epresents  opsonic  indices.  A,  skin  grafts  to  arm 
and  leg;  B,  develop raei^^  demonstrated  M.  aureus.  The  small  crosses  on 
the  diagonal  line  indicat°°*^***°°  *°^  health  of  the  patient.  Note  also  that 
no  new  abscesses  formed 


Fig.  43— Linda  C.     Euros  one-third 

I  dia~gonal  line  indicate  inoculation  with  M.  aureus  bacterin.  205.000.000  bacteria,    *Note  the  parallelism  existing  between  opsonic 
new  absoessea  formed  after  the  first  inoculation  of  bacterin  and  the  rapidity  of  disappearance  of  the  recurrent  preftxiatent  aubcutaneous 


BACTERIAL  INOCULATION 


263 


Ulcers, — A  variety  of  bacteria  have  been  identi- 
fied,  the  majority  of  which  in  chronic  and  intractable 


OPSONIC - 

TEIMPERATURE  CHART 

— 



0) 

- 

<  u 

a. 

O 

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<j 

3 

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11 

t 

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1              i^iX'i^Sk 

1 

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~,"^ 

- 

^         1         20 

21 

22 

n 

?A 

5 

'6                 27 

'6 

Hour         I 

5^ 

=t 

ft 

2.!Q:I2 

^IfilQ 

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SQ 

q 

1? 

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6 

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1 

f,c 

5q 

1? 

3 

6 

q 

0 

1? 

>  6  99  12 

^6991 

•)  6 

^T~ 

2 

3 

ef<: 

27- 
P,6- 
25- 
24- 
23- 
2Z- 
2.1- 
20- 
1.9- 
1.6- 
17- 
1.6- 
15- 
14- 
13- 
12- 
11- 
10- 
B- 

s- 

7- 
6- 
5- 
4- 
3- 
2- 

105- 
104- 
103- 
102- 
101  - 

■ 

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20 

12 

2?|2«iJ?|zc|Zl)i20!J0|l5il0|l8i2O-,2:,Z2;?e|l8|2O 

l8|70l25jZOiZo|j 

W..«ht 

IM                                                                                                                                                                                                                                                    If 

Fig.  42. — In  this  case  of  typhoid  fever  a  few  days  after  the  return  of  the  tempera- 
ture toinormal  during  convalescence,  dozens  of  subcutaneous  abscesses  formed  persis- 
tently for  a  week  or  more,  requiring  daily  incisions  and  drainage.  Cultures  demonstrated 
M.  aureus  in  pure  state.  Observe  the  immediate  effect  of  a  single  inoculation  of 
100,000,000  staphylococci.  Coincidently  with  the  fall  in  temperature  and  general 
improvement,  the  opsonic  index  showed  a  marked  rise. 

cases  arise  as  secondary  infections,  against  which  bac- 
terins  may  possess  Httle  value  unless  the  primary  in- 


S64 


APPLIED  IMMUNOLOGY 


fection,  as  tuberculosis,  syphilis,  etc.,  can  be  influ- 
enced. In  addition  to  all  of  the  common  pyogenic 
bacteria,  the  following  should  receive  consideration: 
B.  fusiformis,  B.  punctatum  and  certain  corynebac- 


INDEX 

?  'i^ 

FEB  FEB       MAR      MAR   APR            APR                               MAYI 
18    25         10        Z2      1               IT                                23  1 

(5^ 

T 

1  1 

— g- 

l§    s       §     M 

"""  § 

1  g  1  §      §     ° 

s 

1 5  "1  s      s     s" 

® ^ 

/                            "^""-^^ 

/ 

\                f 

j        \              j/. 

1        1              f 

1         1             / 

1         (»           / 

I         ^          / 

,i.n 

1           \        / 

n.q 

w                   \           / 

n.x 

\      / 

n.T 

\    / 

.,«.& 

\/ 

n.5 

tS5 

,t}.4. 

<a=np.snNin  inrfx 

flfS 

y=1Nnr.ULAT10N  f^TRFPTn-RAr.TRRIN 

Fig.  44. — Mrs.  J.  B.  C.  Recurrent  erysipelas.  Observe  in  this  case  that  the 
"high  tide  of  immunity,"  indicated  by  the  opsonic  indices,  has  been  maintained  for 
two  and  a  half  months,  no  inoculation  having  been  given  since  March  22.  It  will 
be  noted  that  this  is  entirely  in  accord  with  the  clinical  symptoms  of  the  case. 

teria.  In  the  treatment  of  rapidly  spreading  phage- 
denic ulcerations,  excellent  results  have  been  obtained. 
Naturally,  autogenous  preparations  should  be  utilized 
whenever  possible,  and  cultures  repeated  frequently 
as  the  bacterial  flora  are  subject  to  change. 


BACTERIAL  INOCULATION 


265 


Cellulitis  and  Dermatitis. — Under  these  headings 
are  included  erysipelas,  Ludwig's  angina  and  pemphi- 
gus,  commonly  associated  with  lymphangitis  and 
lymphadenitis.  The  preponderant  invading  organism 
is  the  streptococcus,  although  the  staphylococcus  is 
also  found.     Streptococcal  infections  of  this  class,  in 


INDEX         Ml                    19  Z     1    1    1                                                                            9   3 
NOV,                            pec,                                                                    JU^Y 

li Zfebbh  )0  1  2.i£l(LliIllQlllilil4  4  5  t  7  8  l1£niililllllilIliiayiil^aJ4^Lfc2,n8Z2  30  51 

A. 5            III                              l_L...                          - 

x=  iNonii.ATinw  STR  prncqcci                                         _        _             _ 

Jh ^ 

J  J\                           ------- 

^-     \ 

S 

■^"^^c'c                    -c-                         --=                1        i                      [        g           5 _        i 

^i  T  "■  '  %             ItIB          ak                § 

3""l'  ■       1               §      1  3      /    a      ^                 ^ 

g=                °                       ^"=?Zi 

?..     s  -  i               i                      s          - "  s "       '^       s            ^                       S 

:      :/         :     :\         :        _ 

t               ^ 

::      §    ~  " 

i,fi    -                                   __7           __      -^__  _     _  _ 

f--                        ^                               -■;; 

~_J5. s,              "                                                        I                                    ~                                             ^ts'^ 

-U-.^ ^-,____^^ .,-,.- - 

i^     "                        '^   -^^^^           -     -     -- 

r 

.5              ,      „X ,       - 

Fig.  45. — Mrs.  C.  Tonsillitis,  peritonsillitis  and'toxic  arthritis.  Even  after  deep 
cervical  incisions  through  an  extensive  cellulitis  to  the  tonsillar  region  for  drainage, 
the  patient  did  not  progress  satisfactorily  until  the  administration  of  an  autogenous 
bacterin,  which  exerted  a  specific  and  immediate  beneficial  effect  both  on  the  cervical 
infection  and  also  on  the  arthritic  manifestations. 

the  acute  stage,  run  a  short  course  immeasurably  in- 
fluenced by  bacterins;  in  the  subacute,  chronic  or  re- 
current stages,  bacterial  inoculations  have  proved  of 
immense  benefit,  at  times  performing  the  incredible 
(Figs.  44  and  45). 

Impetigo   and  Sycosis, — The   invading  microbe 


S66 


APPLIED  IMMUNOLOGY 


may  be  either  the  staphylococcus  or  streptococcus, 
consequently  culture  and  the  appropriate  autogenous 
bacterin  may  be  employed  with  benefit.  In  impetigo 
the  result  is  uniformly  successful ;  in  sycosis  treatment, 
ofttimes  prolonged,  should  result  in  fifty  per  cent, 
cures,  the  remainder  showing  improvement. 

Sinus    and    Fistula. — Various    bacteria,    as    the 
Staphylococcus,  Streptococcus,  B.  coli,  B,  proteus  vul- 


Fia.  46. — Temperature.  Patient,  W.  F.  Subdiaphragmatic  abscess  drained  per 
laparotomy;  a,  patient  very  comfortable,  drainage  satisfactorj';  h,  patient  complains 
of  pain  in  hepatic  region;  c,  leucocytes  20,000;  d,  excessive  suppuration;  e,  suppura- 
tion variable  but  very  profuse;  /,  patient's  appearance  improved,  feels  much  stronger; 
g,  patient  discharged  from  ward;  h,  treated  in  out-patient  department;  i,  suppuration 
markedly  decreased;  k,  discharge  from  wound  very  slight;  /,  marked  gain  in  strength 
and  health;  m,  no  discharge;  n,  sinus  healed;  1,  pus  demonstrates  M.  aureus  and 
alhus;  2,  inoculations  M.  aureus  bacterin  J^  c.c.  This  case  illustrates  how  admirably 
active  immunization  can  be  conducted  by  resort  to  the  clinical  symptomatology  as 
a  guide,  to  the  exclusion  of  the  opsonic  index. 

garis,  B,  tuberculosis,  B,  pyocyaneus,  B.  fluorescens, 
B,  lactis  aerogenes,  etc.,  have  been  found.  The  re- 
sults in  fistulse  have  been  unsatisfactory;  in  sinuses,  if 
no  surgical  indications  exist  and  lavage  of  the  tract 
with  Wright's  solution  to  promote  osmosis  and  hyper- 
semia  is  used  to  facilitate  the  effectiveness  of  therapy. 


BACTERUL  INOCULATION 


267 


bacterins  and  tuberculins  in  many  cases  have  possessed 
considerable  accessory  value  (Figs.  46  and  47).  Our 
experience  with  pyocyaneus,  fluorescens,  and  lactis 
aerogenes  bacterins  in  any  condition  has  never  been 
glorifying.  Tuberculosis  commonly  involves  the  skin 
and  subcutaneous  tissues,  especially  the  lymph-nodes, 
and  is  notably  associated  with  sinuses  and  fistul^e. 


m 

10S 

m 

101 
100 
93 
il 
9T 
91 
95 
9+ 

INDEX 

AUG" 

Z  3  4-  ! 

\ 

T 

8 

9  HO 

3 

II 

'■ 

P 

T 

i4 

E 

MB 

-R 

319 

zo 

Z1 

u 

J5 

M 

a 

r 

- 

:e 

:9 

" 

: 

1 

J 

4' 

0 

5 

C 

T 
T 

0 

B 
9 

E 

10 

R 

11 

li 

1114  IS 

®=  OPSONIC  INDEX 

X=  INOCULATIOMS    OF  BACTERIA 

y 

- 

<^ 

'~^ 

L-J 

T • 1PERATURE 

' 

/ 

■^ 

if^ 

J 

/ 

, 

I7t 

^ 

^■** 

* 

•t 

^" 

-  ■  W 

', 

J^ 

-^ 

. 

^ 

" 

L_ 

^ 

^ 

' 

^ 

f 

^  '^ 

'.        s 

/» 

» 

« 

^ 

^     ■** 

y 

' 

«' 

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, . 

Vf 

.^ 

'' 

■* 

• 

•  ^ 

' 

~"? 

g 

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%~ 

^ 

s 

1 

=  1 

^ 

fe 

2 

t 

:^ 

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~    "    0= 

'^ 

i? 

£ 

3 

^__ 

-= 

S 

- 

a. 

■XL 

S 

- 

J 

_^ 

_ 

_ 

_ 

Fig.  47. — Mrs.  B.    Pelvic  abscess  with  recto-urethro-vaginal  fistulae.     Note  the  fall  of 
temperature  to  normal  as  the  curve  of  opsonic  antibodies  rises. 

The  reader  is  referred  to  Chapter  XYI,  where  the  in- 
oculation treatment  of  this  disease  is  detailed. 

Actinomycosis. — Actinomycoses  may  involve  the 
lungs,  liver,  kidneys,  bladder,  brain,  abdominal  viscera, 
bones,  genitalia  or  subcutaneous  tissues.  Actinomyco- 
tic inoculations  in  the  pulmonary,  hepatic,  renal, 
vesical  and  cerebral  forms  of  the  disease  are  of  little 
or  no  value.  Actinomycotin,  standardized  so  that  one 
cubic  centimetre  contained  one  milligramme  of  the 


268  APPLIED  IMMUNOLOGY 

sterilized  powdered  organisms,  we  have  found  bene- 
ficial in  superficial  localized  lesions,  using  an  initial 
dose  of  0.001  milligramme. 

Glanders. — Bacterins  of  B.  mallei  or  "  mallein  " 
have  been  attended  with  indifferent  success  and  in  a 
generalized  infection  of  glanders  offer  no  promise  of 
cure  whatsoever. 

Variola. — Smallpox,  the  greatest  and  most  loath- 
some scourge  that  ever  decimated  the  human  race,  is 
to-day  a  disease  of  comparative  rarity.  With  an  inci- 
dence of  ninety-five  per  cent,  among  the  exposed  and 
a  mortality  of  twenty  per  cent.,  in  the  eighteenth  cen- 
tury it  was  estimated  that  2,000,000  people  in  Europe 
annually  contracted  variola,  of  whom  400,000  died. 
Jenner's  vaccination  against  smallpox  in  1798— the 
first  and  classical  example  of  the  prophylactic  em- 
ployment of  bacterial  inoculation — has  resulted  al- 
most in  the  extermination  of  this  disease.  Physicians 
of  eminence  in  every  civilized  country  of  the  globe 
fully  realize  the  inestimable  value  of  this  achievement 
for  mankind  and  appreciate  the  fact  that  vaccinia 
properly  employed  protects  against  variola.  In  Ger- 
many, where  the  most  advanced  and  adequate  legis- 
lation has  been  enacted  for  forty  years,  compulsory 
vaccination  and  revaccination  have  rendered  security 
against  smallpox  almost  absolute.  Originally  crusts 
from  variola  ulcers,  then  crusts  from  the  vaccinia 


BACTERIAL  INOCULATION  269 

sores  of  othei-wise  healthy  individuals,  were  utilized. 
Later  experiments  on  calves  showed  that  smallpox 
virus  can  be  converted  into  vaccine  virus  by  passage 
through  several  (four)  generations  of  the  bovine 
species.  Thus  the  virus  is  rid  of  any  possible  con- 
tamination from  human  sources  and  can  be  and  is  pre- 
pared in  any  desired  quantities.  The  best  vaccine 
virus  is  glycerinized  and  a  number  of  reliable  pharma- 
ceutical firms  market  the  lymph  in  sealed  capillary 
glass  tubes,  protected  vaccine  points  and  vials.  The 
virus  must  be  preserved  in  a  refrigerator. 

Technic  of  Vaccination, — The  skin  of  the  arm  pref- 
erably or  the  leg  is  cleansed  with  alcohol  or  ether. 
The  superficial  layer  of  the  epidermis  is  denuded  by 
a  sterile  von  Pirquet  scarifier  or  sharp  dental  chisel 
by  a  rotary  motion,  or  in  the  absence  of  these  by  a 
scalpel  or  needle,  producing  an  area  about  two  milli- 
metres in  diameter,  caution  being  observed  to  draw 
only  serum,  not  blood.  The  scarification  may  be  done 
through  a  drop  of  virus  primarily  placed  on  the  skin 
or  the  virus  may  be  deposited  when  the  denudation  is 
completed  and  rubbed  in  with  a  sterile  orange  stick 
or  toothpick.  If  possible  the  di'op  of  lymph  should 
be  allowed  to  dry  in  the  air  or  after  fifteen  minutes  a 
gauze  dressing  or  vaccine  shield  may  be  applied  to  the 
inoculated  site  for  protection.  The  cow-pox  wound 
should  be  inspected  daily  for  at  least  fixe  days. 


270  APPLIED  IMMUNOLOGY 

Characteristics  of  Vaccinia  Sites. — Due  to  trauma 
alone,  a  few  minutes  after  vaccination  the  skin  im- 
mediately surrounding  the  wound  assumes  a  reddish 
line ;  this  disappears  in  a  few  hours.  If  at  the  end  of 
twenty-four  hours  the  vaccinated  spot  exhibits  an 
areola,  with  or  without  papule,  one-half  centimetre  or 
more  in  diameter,  which  inflammatory  zone  decreases 
in  seventy-two  hours,  it  is  to  be  regarded  as  an  al- 
lergic reaction  of  immunity  due  to  specific  antibodies 
in  the  individual  and  excuses  him  from  fm'ther  im- 
mediate vaccination.  If  the  areola  observed  at  the  end 
of  twenty-four  hours  develops  into  a  small  vesicle 
maturing  on  the  fifth  or  sixth  day,  then  rapidly  sub- 
siding, it  is  to  be  regarded  as  a  case  of  vaccinoid,  that 
is,  one  where,  although  antibodies  are  not  present,  the 
power  to  form  them  exists  from  previous  vaccination. 
If  reactionary  changes,  characterized  by  a  reddened, 
inflammatory,  indurated  areola  or  aula  surrounding 
a  papule,  supervened  only  from  the  second  to  the  fifth 
day,  followed  by  flattening  of  the  smnmit  of  the 
papule,  it  is  to  be  regarded  as  a  case  of  typical  vac- 
cinia.  By  the  tenth  day  the  reaction  will  have  reached 
its  maximum,  the  inflammatory  areola  measuring 
sometimes  more  than  five  centimetres  and  the  papule 
as  much  as  one  centimetre.  The  papilla  at  the  onset 
assumes  a  pale  pinkish-gray  color,  changing  to  a  gray- 


BACTERIAL  INOCULATION  271 

ish-yellow,  followed  by  rapid  drying  and  crust  forma- 
tion. Involution  begins  usually  about  the  tenth  day 
and  by  the  end  of  the  second  week  only  a  thick,  brown, 
tightly  adlierent  crust  surrounded  by  pigmented  skin 
marks  the  original  site  of  the  papule. 

From  the  standpoint  of  therapy  it  is  of  interest  to 
note  that  recently  autogenous  staphylococco-bacterins 
prepared  from  the  pustules  of  smallpox  have  been 
successfully  used  in  diminishing  the  degree  of  cutane- 
ous pitting. 

Bubonic  Plague, — Treatment  with  bacterial  sus- 
pensions of  B.  pestis  has  been  extensively  practiced 
by  HafFkine.  Prophylactic  inoculation  is  of  much 
greater  value  than  therapeutic  administration,  and 
has  reduced  the  mortality  from  66.6  to  16.6  per  cent, 
in  the  experience  of  Strong,  who  employed  living 
attenuated  bacilli.  The  primary  immunizing  dose 
should  be  five  billion  bacteria,  followed  in  ten  days  by 
ten  billions.  Immunity  is  alleged  to  endure  from 
three  to  eighteen  months.  Therapeutic  inoculation 
greatly  reduces  the  severity  of  attacks,  and  recovery 
always  occurs  if  the  victim  be  a  European.  Cura- 
tively,  however,  Yersin's  or  Lustig's  antitoxic  serum 
supersedes  bacterin  as  a  therapeutic  measure.  Bac- 
terins  are  probably  valueless  in  the  septiceemic  and 
pneumonic  forms  of  the  disease. 


APPLIED  IMMUNOLOGY 


M  ^  *«  M  ^  w.  ^  »^  I-' _>.  ;s>  ro  ^»  rs>  zs>  e^  t^  to  («> 


DMcHtirje  freer.  temperi&TB   (|j\ 
sUjWly  lower.  1^ 

5u,p  p  uT  a.tvOTt  i  jcr  e  a  se  i. 


Free  ilsckir^e  of  pos, 
temp.fell  \y  crisis  from 
105°  to  gg',  tumor  maa* 
smiUer. 

Sappuration.  decreasel 
teaierness  over'  kiiney 
is  less  , 

Temjera.ture  rea.thesio4. 
ani  IS  intermittent. 

Tem,peratu.Te  lower,  not 
over  103". 


Citric   a.cld  ititernaUy. 


Kiiney  mass  smaller 


Dlsclvar^e  scajity  and. 
watery. 

Temperature  fluctuates 
between  gf*  and  103°. 

Markei  improvement. 


P.  improved  oeneraUy. 
Fijtuia  iressei  temi-weekly. 


P.stron^er,  ooes  to  jeasKori 
to  recuperate. 
Kiiney  mass  sXrinkm^ 
away. Temperature  reatlves 
100*  16101°  in  evening 


Returns  from  sea 
oatti  In  weiont.  2,3 1 
Vistulj  '      ^       ' 


FisXula  barely  aimiTsjTO 
CUcharje  scanty  ft  Sterile . 


Fig  48  —Dorothy  W.  Pyonephrosis.  Nephrectomy  in  this  case  was  impossible 
owing  to  the  size  and  adherent  state  of  the  kidney.  Nephrectomy  temporarily  relieved 
for  a  few  months;  then  the  patient  became  critically  ill  and  was  rescued  apparently 
by  autogenous  bacterin  therapy.  Note  the  unprecedented  high  flights  of  the  opsomo 
index,  especially  that  of  September  14. 


BACTERIAL  INOCULATION 


273 


Diseases  of  the  Genito-urinary  System 
Cystitis,  Pyelitis,  Pyelonephritis  and  Pyonephro- 
sis.— Bacterins  from  the  following  microorganisms 
have  been  of  service  in  certain  diseases  of  the  kidney 
and  bladder;  B,  coli,  B.  tuberculosis,  B,  pseudotuber- 
culosis rodentium,  B,  of  Friedldnder,  B.  typhosus. 


<Z  JxLr                 27             3               10  Auo.    17              ZS           31 

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Fig.  49.— Albert  P.  Reno-lumbar  fistula  following  nephrolithotomy  complicated 
by  pyonephrosis.  Note  occurrence  of  a  mixed  infection  in  August,  demonstrating  the 
advisability  of  autogenous  bacterins  and  the  necessity  of  re-culture  at  intervals.  > 

=inoculation  with  colon  bacilli; >  =  inoculations  with  streptococci. 

Streptococcus  pyogenes.  Staphylococcus  and  M.  lan- 
ceolatus.  Great  discrimination  must  be  exercised  as  to 
whether  the  particular  condition  is  amenable  to  bac- 
terin  therapy  or  should  be  operated  upon.  Bacterins 
were  never  intended  to  supplant  surgery,  but  at  times 

18 


274 


APPLIED  IMMUNOLOGY 


are  wonderful  accessory  agents  in  promoting  cure, 
and  not  infrequently,  with  proper  administration,  re- 
lieve surgery  of  some  of  its  indications.  In  colon 
bacillus  infections  of  the  bladder  and  kidney  in  chil- 
dren, also  in  kidney  suppurations  where,  owing  to  the 
size  and  adherent  character  of  the  diseased  organ. 


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nephrectomy  is  temporarily  impossible,  autogenous 
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UretJmtis, — Compared   with   the   gonococcus   of 
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BACTERIAL  INOCULATION 


275 


insignificance,  although  occasionally  a  non-specific  in- 
fection arises,  due  to  the  31,  catarrJialis  or  other  pyo- 
genic bacteria.  Gonococcus  bacterin  has  been  utilized 
both  prophylactically  and  therapeutically  against 
gonorrhoea.  As  a  protective  measure,  Douglas, 
Wright's  associate,  recommends  an  initial  dose  of 
100,000,000.  If  this  is  followed  by  no  reaction  a 
second  inoculation  of  200,000,000  is  administered  in  a 
week  or  ten  days.    The  adoption  of  this  procedure  is 


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Fig.  51. — Cystitia  and  toxic  neuritis. 


advisable,  although  unattended  with  any  absolute  as- 
surance, in  certain  marital  relations  when  the  disease 
is  innocently  acquired,  for  the  protection  of  girls  in  an 
institution  in  the  event  of  an  outbreak  of  gonorrhceal 
vulvovaginitis,  and  perhaps  to  safeguard  innocent 
newlyweds,  under  peculiar  circumstances,  when  one 
of  the  contracting  parties  has  recently  convalesced 
from  an  attack  of  urethritis. 

Again,  it  must  be  borne  in  mind  that  the  discharge 


276  APPLIED  IMMUNOLOGY 

of  an  acute  gonorrhoea,  sooner  or  later,  invariably 
contains  bacteria  other  than  the  gonococcus,  namely, 
the  Streptococcus  pyogenes,  the  Staphylococcus,  the 
Pneumococcus,  the  Colon  bacillus,  the  Micrococcus 
catarrhalis,  etc.  Thus  the  prophylactic  effect  of  early 
immunization  in  a  case  of  gonorrhoea  against  these 
complicating  germs,  in  the  prevention  or  ameliora- 
tion of  such  conditions  as  gleet,  prostatitis,  seminal 
vesiculitis,  etc.,  should  not  be  underestimated.  The 
authors  feel  that  in  many  cases  in  their  experience  the 
cautious  administration  of  mixed  gonococcic  bacterin 
has  sufficed  to  shorten  convalescence  and  to  prevent  or 
reduce  the  severity  of  certain  complications,  notably 
inflammation  of  the  prostate  and  seminal  vesicles  and 
stricture  formation.  It  is  a  difficult  or  impossible  task 
to  gauge  the  effect  of  treatment  upon  any  disease 
prone  to  run  a  definite  course,  consequently  the  value 
of  bacterins  in  urethritis,  in  nmnberless  instances,  has 
been  immeasurable,  and  although  certain  investiga- 
tors have  reported  favorably  on  biological  therapeusis 
in  acute  gonorrhoea,  consensus  of  opinion  discourages 
the  procedure. 

In  this  connection  reference  must  be  made  to 
Bruck's  "  Arthigon,"  a  suspension  of  dead  gonococci, 
standardized  to  contain  twenty  millions  per  cubic 
centimetre.  Trustworthy  evidence  has  accumulated 
to  the  effect  that  the  intravenous  injection  of  this 


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BACTERIAL  INOCULATION  277 

preparation  in  quantities  of  0.05  to  0.5  c.c.  produces  a 
reaction  which  is  invariably  constant  in  gonorrhoeal 
subjects.  A  rise  of  temperature  above  1.5°  C.  after 
an  injection,  of  1  c.c.  may  be  regarded  as  pathogno- 
monic. Therapeutically,  with  increasing  dosage,  the  re- 
sults have  been  no  better  than  with  the  subcutaneous 
administration  of  bacterins  or  sero-bacterins,  and  it  is 
extremely  doubtful  if  intravenous  injection  will  super- 
sede the  older  method,  properly  interpreted,  in  a  diag- 
nostic capacity. 

Prostatitis  and  Seminal  Vesiculitis, — The  writers 
believe  that  these  complications,  in  addition  to  the 
gonococcus,  are  commonly  precipitated  and  perpetu- 
ated by  a  mixed  infection  due  to  the  Streptococcus, 
PneumococcuSj  Colon  bacillus,  M,  catarrhalis,  etc. 
They  have  obtained  in  many  instances  gratifying  re- 
sults by  alternating  a  decivalent  gonococcus  stock  and 
autogenous  bacterins,  the  latter  prepared  from  urine 
cultures  after  massage  of  prostate  and  seminal  vesi- 
cles, when,  in  addition  to  the  above,  other  organisms, 
as  the  Micrococcus  alhus,  aureus,  citreus,  candicans, 
Candidas  and  orbicularis,  the  Bacillus  typhosus,  pyo- 
cyaneus  and  acidi  lactici  and  the  Cory  neb  acterium 
pseudodiphtheriticum,  have  been  isolated.  The 
therapy  has  been  conducted  only  in  the  subacute  and 
chronic  cases,  preferably  in  the  presence  of  suppura- 
tive inflammations  ( Fig.  52 ) . 


278 


APPLIED  IMMUNOLOGY 


Epididymitis, — Gonococcus  bacterin  is  probably 
of  value  in  acute  epididymitis  (Fig.  53),  although 
the  disease  is  prone  to  run  a  definite  short  course  under 
proper  treatment  without  immunotherapy.  It  would 
appear,  however,  that  the  employment  of  bacterin  di- 


102 


99 


98 


% 


10    11    \z 
MEMEME 


l\ 


w 


w 


a 


Fig.  53. — Typhoid  fever  complicated  by  epididymitis.  Observe  transient  slight 
fall  in  temperature  a  few  days  after  first  inoculation  of  typho-bacterin,  and  the  fall 
by  lysis  after  the  second  dose  of  bacterin. 


minishes  the  likelihood  of  a  resultant  inflammatory 
nodule  of  the  epididymis,  at  the  same  time  reducing 
the  liability  to  sterility.  In  the  chronic  form  due  to 
tuberculosis,  tuberculin  is  of  unquestionable  value, 
both  diagnostically  and  therapeutically,  although  in 
no  small  numbers  of  cases  epididymectomy  must  be 


BACTERUL  INOCULATION  279 

performed,  and  this  applies  particularly  to  dispensary 
patients. 

Vulvovaginitis, — In  children  inoculations  of  a 
polyvalent  gonococcus  bacterin,  in  the  acute  stage, 
have  resulted  brilliantly,  causing  the  early  disappear- 
ance of  gonococci  and  shortening  the  usual  course  of 
treatment  by  months.  Five  million  gonococci  consti- 
tute the  average  dose.  From  three  to  a  dozen  inocula- 
tions are  required.  Benefit  is  also  observed  from  autog- 
enous bacterins  in  the  chronic  form  of  the  disease 
due  to  other  pyogenic  bacteria,  as  the  Streptococcus, 
Staphylococcus,  Pneumococcus  and  Colon  bacillus. 
Vaginitis  in  adults,  in  the  experience  of  the  authors, 
has  been  absolutely  uninfluenced  by  bacterial  inocula- 
tions. 

Cervicitis,  Endometritis,  Metritis  and  Salpingitis, 
— The  following  bacteria  have  been  isolated:  31, 
gonorrhoece,  B,  coli,  Pneumococcus,  Streptococcus, 
Staphylococcus  and  Tubercle  bacillus.  This  category 
of  diseases  needs  further  investigation  respecting  the 
value  of  bacterins,  since  the  studies  to  date  have  been 
inconclusive.  In  general  the  results  have  been  very 
indifferent,  scarcely  warranting  the  procedure.  This 
is  certainly  true  in  the  case  of  cervicitis.  In  the  treat- 
ment of  salpingitis  a  number  of  remarkable  results 
have  been  reported.    An  essential  to  success  lies  in  the 


280  APPLIED  IMMUNOLOGY 

selection  or  preparation  of  the  appropriate  bacterin, 
dependent  upon  the  determination  of  the  invading 
microbe. 

Puerperal  sepsis  is  entitled  to  special  and  serious 
consideration.  In  our  opinion  the  applicability  of 
bacterins  is  directly  dependent  upon  the  presence  or 
absence  of  a  general  bacteremia,  determinable  by 
blood  culture.  If  the  former  exists,  active  immuniza- 
tion is  definitely  contra-indicated ;  if  the  septic  process 
is  localized  to  the  pelvis,  bacterial  inoculation,  cau- 
tiously employed,  may  prove  serviceable.  We  have 
been  gratified  on  several  occasions  by  the  excellent 
results  of  bacterins  supplementing  or  alternating 
passive  immunization  (serum  therapy).  In  any 
event,  this  is  a  condition  wherein  opsonic  indices  will 
render  important  service  in  the  control  of  therapy. 

Diseases  of  Boxes  and  Joints 

Osteitis,  Periostitis  and  Osteomyelitis, — Infections 
from  the  following  bacteria  come  into  consideration: 
Staphylococcus,  Streptococcus,  Pneumococcus,  Gono- 
coccus.  Typhoid  hacillus.  Colon  bacillus  and  Tubercle 
bacillus.  In  the  acute  stage  of  an  osteoperiostitis, 
if  bactersemia  is  not  present,  bacterin  therapy  is  very 
beneficial,  especially  in  pneumococcic,  gonococcic, 
typhoid  and  colon  infections ;  in  early  spur  formation 


BACTERIAL  INOCULATION 


281 


from  the  os  calcis  (osteophytes,  exostoses)  the  ad- 
ministration of  gonococcus  bacterin  has  rendered 
operative  intervention  unnecessary,  or  at  least  has 
eliminated  toxins  and  insured  a  more  satisfactory 
post-operative  result.    In  chronic  cases  it  is  even  more 


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arrow  indicated  when  a  blood  culture  demonstrated  M.  aureus;  the  crosses  represent 
the  times  of  inoculations  with  M.  aureus  bacterin.  Observe  that  the  patient,  as  indi- 
cated by  the  increased  fluctuations  of  temperature,  progressively  grew  more  septic, 
and  that  bacterin  therapy  was  not  only  useless,  but  possibly  harmful.  The  substitution 
of  bacterin  at  the  juncture  by  antistaphylococcic  serum  apparently  exerted  a  definite 
beneficial  influence  for  recovery. 

meritorious,  if  caries  and  sequestra  are  excluded  and 
hypergemia  used  to  augment  the  treatment.  In  our 
experience  bacterins  and  tuberculins  have  done  more 
harm  than  good  in  acute  osteomyelitis  (Fig.  54). 

Synovitis  and  Arthritis, — Associated  with  these 
diseases  the  Streptococcus  rheumaticus,  Gonococcus, 


282  APPLIED  IMMUNOLOGY 

Pneumococcus,  Streptococcus  pyogenes  and  Staphy- 
lococcus should  be  mentioned.  Bacterins  compiled 
from  the  first-named  organism,  whether  isolated  from 
the  joint,  gmns,  tonsils  or  feeces,  have  in  a  number 
of  instances  rendered  therapeutic  service  in  the  acute, 
subacute  and  chronic  forms  of  disease.  We  have  never 
observed  any  remarkable  benefit  by  the  employment 
of  the  common  pyogenic  streptococci  and  staphylo- 
cocci, nor  is  much  to  be  expected  in  chronic  cases 
where  hypertrophic  osseous  changes  have  occurred. 
On  the  other  hand,  this  is  a  field  wherein  gonococcus 
bacterin  enjoys  much  distinction.  Indeed,  biological 
inoculations  furnish  the  treatment  pai'  eoccellence  for 
gonorrhoeal  synovitis  and  arthritis.  Many  patients, 
with  limitation  of  motion  in  subacute  and  chronic 
gonorrhoeal  joints,  are  best  treated  by  preliminary 
inoculations  of  Neisser  bacterin  a  few  weeks  or  months 
prior  to  forcible  manipulation  under  general  anaes- 
thesia, thereby  eradicating  the  cause  of  the  inflam- 
matory exudate  and  preventing  its  re-formation  after 
operation. 

It  should  also  be  observed  in  this  connection  that 
gonococcus  bacterin  is  of  exceptional  value  in  the 
differential  diagnosis  of  many  obscure  joint  and  other 
gonorrhoeal  afflictions,  used  precisely  as  is  tuberculin 
in  a  diagnostic  capacity. 


BACTERL\L  INOCULATION  283 

Diseases  of  the  Eye,  Ear,  Nose  and  Throat 
Singularly  perhaps,  but  nevertheless  fortunately, 
infections  of  the  eyes  have,  in  the  majority  of  in- 
stances, demonstrated  the  great  value  of  bacterin 
therapy ;  not  so  much  can  be  claimed  in  the  treatment 
of  diseases  of  the  ear,  nose  and  throat.  There  are 
certain  anatomical  facts  explanatory,  at  least  in  the 
case  of  the  ear,  of  these  variable  effects. 

Conjunctivitis  and  Dacryocystitis, — The  follow- 
ing bacteria  have  been  isolated:  Pneumococcus, 
Streptococcus,  Staphylococcus,  Gonococcus,  M,  catar- 
rhalis,  Friedldndefs  bacillus,  Koch-Weeks  bacillus, 
Moraoj-Accenfeld  bacillus,  Pyocyaneus  bacillus  and 
Tubercle  bacillus.  In  many  acute  infections,  notably 
that  due  to  the  bacillus  of  Koch- Weeks,  the  usual 
ophthalmological  treatment  will  prove  all-sufficient, 
while  in  chronic  types,  as  that  caused  by  the  bacillus 
of  Morax-Axenfeld,  frequently  little  response  fol- 
lows ordinary  treatment  and  the  effect  of  bacterins 
appears  to  be  specific.  Between  these  are  a  number 
of  infections  due  to  the  gonococcus,  pneumococcus, 
streptococcus,  etc.,  which  may  do  well  with  ordinary 
measures,  but  in  which  the  accessory  employment  of 
bacterins  hastens  convalescence,  diminishes  the  in- 
roads of  the  pathological  process,  prevents  sympa- 
thetic ophthalmia  and  avoids  the  loss  of  vision.     In 


284  APPLIED  IMMUNOLOGY 

dacryocystitis  bacterin  therapy  may  obviate  surgical 
extirpation  of  the  lachrymal  sac. 

Corneal  Ulcer  and  Hypopyon. — The  Diplococcus 
pneumonke,  the  Streptococcus  mucosus,  the  Strepto- 
coccus pyogenes,  the  Staphylococcus,  the  Gonococcus 
and  Tubercle  bacillus  have  been  identified.  Autog- 
enous bacterins  prepared  from  the  pneumococcus 
and  streptococcus  in  cases  of  ulcus  serpens  of  the 
cornea,  with  and  without  hypopyon,  notably  in  one 
instance  in  which  panophthalmitis  threatened  com- 
plete destruction  of  the  eye,  have  produced  incredible 
results  (Fig.  55).  Care  must  be  observed  not  to 
employ  too  small  dosage  in  the  treatment  of  these 
affections.  The  initial  inoculation  should  be  at  least 
100,000,000  bacteria. 

Iritis  and  Uveitis. — Here  as  in  corneal  ulcer  the 
same  organisms  are  found  at  work.  If  the  infecting 
bacterium  be  determinable  the  results  are  invariably 
excellent.  Especially  noteworthy  is  bacterin  therapy 
in  gonorrhceal  involvements.  In  these  conditions,  as 
in  practically  all  gonococcic  complications,  practi- 
cability demands  that  a  polyvalent  stock  preparation 
be  employed. 

Otitis  Media  and  Mastoiditis. — An  extensive  array 
of  bacteria  as  the  Streptococcus,  Staphylococcus^ 
Pneumococcus,  Colon  bacillus,  B.  proteus  vidgaris, 
B.  lactis  aerogenes,  B.  pyocyaneus,  B.  fluorescens,  B, 


BACTERIAL  INOCULATION 


285 


diplitlience  and  pseudodiphtherice,  31.  catarrhalis,  B. 
influenzce,  B,  typhosus  and  B,  tuberculosis  have  been 
found.  Due  to  the  fact  that  in  the  majority  of  cases 
the  cultured  organism  is  a  secondary  invader,   also 


JANUARY 
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FiG.  55. — V.  S.  Corneal  ulcer  with  hypopyon.  Culture  of  pus  from  hj-popyon 
demonstrated  the  Streptococcus  mucosus,  from  which  an  autogenous  bacterin  was 
prepared.  We  believe  that  this  patient  was  over-treated,  that  is,  inoculated  at  too 
short  intervals.  Although  he  made  a  satisfactory  recovery,  it  is  probable  that  the  result 
would  have  been  more  brilUant  with  less  frequent  inoculations. 

that  the  infected  area  is  encased  by  bony  walls  limit- 
ing the  supply  of  blood,  although  highly  opsonized, 
the  results  of  bacterin  therapy  have  been  poor,  espe- 
cially in  chronic  cases.  Nevertheless,  in  the  early  stage 
of  otitis  media  complicating  scarlet  fever,  the  sup- 


286  APPLIED  IMMUNOLOGY 

puration  seems  to  be  markedly  curtailed  and  convales- 
cence proportionately  shortened.  On  the  other  hand, 
tuberculin  in  carefully  selected  cases  of  ear  and  mas- 
toid tuberculosis  has  proved  of  great  benefit. 

Rhinitis  and  Sinusitis. — The  bacterial  flora  of  the 
nasal  passages  and  associated  sinuses  differ  little  from 
those  of  the  auditory  apparatus.  The  results  follow- 
ing the  use  of  bacterins,  however,  are  much  better, 
due  probably  to  the  greater  vascularity  of  the  parts 
affected.  Indeed,  in  suppurative  sinusitis  the  em- 
ployment of  autogenous  bacterins,  aside  from  any 
surgical  indication,  should  constitute  the  sheet-anchor 
in  treatment  (Fig.  56) .  The  chief  offending  bacteria 
are  Streptococci,  Pneumococci  and  Micrococci  catar- 
rhalis.  The  dosage  frequently  must  be  increased  over 
the  average. 

Laryngitis  and  Tracheitis, — The  following  bacteria 
are  commonly  demonstrable:  3/,.  catarrhalis,  D. 
pneumonice.  Streptococcus  pyogenes.  Staphylococcus, 
M,  paratetragenus,  B.  diphtherice,  B,  influenzcB  and 
B,  tuberculosis.  Occasionally,  excellent  results  have 
attended  the  use  of  bacterins  in  these  affections,  par- 
ticularly with  the  M,  catarrhalis,  Pneumococcus, 
Streptococcus  and  Staphylococcus, 

Diphtheria. — In  recent  years  active  immunization 
against  diphtheria  has  been  studied  extensively  by 
several  observers.    If  those  who  are  naturally  immune^ 


BACTERIAL  INOCULATION 


287 


as  determined  by  the  Schick  reaction  (see  p.  191) ,  are 
eliminated,  the  results  of  active  immunization,  em- 
ploying either  autolyzed  diphtheria  bacilli  themselves 
or  toxin-antitoxin  mixtures,  have  not  been  brilliant 
and  do  not  by  any  means  warrant  its  substitution  for 


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Notes 

AnW,»                                     •J::,Zi                  ,7/              ..crrtlon-              COM                  '.mm',;'           f.^.',^           0.«h.r,«J          J 

Fig.  56. — Cultures  both  from  ethmoidal  sinuses  and  bronchial  expectoration  dem- 
onstrated the  Streptococcus  pyogenes  in  preponderance,  together  with  the  pneumococcus, 
staphylococcus  and  M.  catarrhalis.  _  Opsonic  indices  were  taken  only  for  the  strepto- 
coccus, although  therapy  waa  carried  out  by  inoculations  with  the  autogenous  mixed 
bacterin. 

antitoxin.  In  the  first  place,  the  formation  of  pro- 
tective antibodies  is  slow,  seldom  before  the  second 
week,  hence  the  superiority  of  immune  serum  in  epi- 
demics, both  prophylactically  and  therapeutically. 
Park  and  Zingher  point  out  that  there  is  lack  of  a 
sufficient  response  to  active  immunization  in  at  least 


288  APPLIED  IMMUNOLOGY 

fifty  per  cent,  of  patients  susceptible  to  diphtheria 
and  that  the  immunity  conferred  lasts  not  more  than  a 
year  or  two.  An  important  conclusion  of  their  work 
is  that  the  Schick  test  will  demonstrate  the  futility 
of  immunization  in  approximately  two-thirds  of  those 
exposed  to  diphtheria. 

Respecting  the  value  of  treatment  of  so-called 
"  diphtheria  carriers  "  by  bacterins,  opinion  is  divided. 
Some  remarkable  results  have  been  reported  and  the 
procedure  deserves  further  trial  before  conclusions 
are  drawn. 

Scarlet  Fever, — Opinion  has  grown  to  the  effect 
that  if  the  streptococcus  is  not  the  cause  of,  it  is  ma- 
terially concerned  in  the  pathogenicity  of  the  com- 
plications of  scarlatina.  Experience  has  shown  that 
bacterins  of  the  Streptococcus  conglomeratus,  what- 
ever may  be  their  effect  curatively  in  the  acute  course 
of  the  disease,  have  unquestionably  favorably  in- 
fluenced the  complications,  as  angina,  suppurative 
rhinitis,  lymphadenitis,  nephritis,  otitis,  mastoiditis 
and  arthritis.  INIost  remarkable  are  the  prophylactic 
inoculations  used  by  Gabritchewsky  in  Russia,  where 
in  thousands  of  cases  specific  immunity  has  been  se- 
cured on  the  average  for  eighteen  months.  Donilow 
states  that  only  1.1  per  cent,  of  inoculated  persons 
contracted  the  disease,  including  those  already  in  the 
incubation   stage;    furthermore,    that   the   mortality 


BACTERIAL  INOCULATION  289 

among  the  inoculated  group  was  only  0.123  per  cent, 
as  compared  with  11.1  per  cent,  among  the  uninocu- 
lated.  For  protective  inoculation  three  doses  of  250, 
500  and  1000  million,  from  a  week  to  ten  days  apart, 
should  be  administered. 

Hay  Fever, — An  allied  treatment  by  antigenic 
inoculations  is  that  of  hay  fever  by  injections  of  the 
extracted  pollen  of  rag-weed,  on  the  assumption  that 
this  affection  is  a  pollen  toxicosis  and  not  an  anaphy- 
lactive  expression,  a  thought  that  has  gained  credence 
recently.  Noon  and  Freeman  gauged  dosage  by  the 
conjunctival  reaction  and  injected  their  patients  at  in- 
tervals of  three  to  ten  days  with  units  representing  the 
amount  of  extract  from  0.001  milligramme  of  pollen. 
Clowes  was  able  to  raise  the  resistance  a  thousandfold, 
although  it  persisted  but  five  months,  by  inoculations 
of  one  cubic  centimetre  of  1  :  5,000,000  to  1  :  500,000 
suspensions. 

Recently,  Lowdermilk  reported  astonishing  re- 
sults with  pollen  toxin,  prepared  as  follows:  One 
gramme  of  mixed  pollen  (250  milligrammes  Ambrosia 
artemisicefolia,  250  milligrammes  Ambrosia  tiifida 
and  500  milligrammes  of  various  varieties  of  soli- 
daco)  was  mixed  with  ten  grammes  of  sterile  sea- 
shore sand  in  a  mortar,  moistened  with  a  part  of 
a  solution  of  100  c.c.  physiological  saline  contain- 
ing   0.5    per  cent,    phenol   and  ground    for    several 

19 


290  APPLIED  IMMUNOLOGY 

hours,  slowly  adding  the  remainder  of  the  saline- 
phenol  solution  until  the  sand  was  reduced  to  an  im- 
palpable powder.  The  whole  was  transferred  to  a 
sterile  flask  and  allowed  to  stand  with  frequent  shak- 
ing, at  room  temperature,  for  twenty-four  hours. 
After  decantation  and  centrifugation  the  toxin  was 
sealed  in  glass  ampoules,  containing  one  cubic  centi- 
metre each.  This  product  was  standardized  so  that  the 
unit  represented  the  quantity  of  toxin  extracted  from 
0.000,001  gramme  of  pollen.  Thus  each  cubic  centi- 
metre contained  ten  thousand  units.  Six  to  eight 
inoculations  were  administered  at  intervals  of  one  to 
ten  days  apart,  representing  from  25  to  1000  units 
each.  The  typical  reactionary  phenomena  simulate 
the  symptoms  of  an  acute  attack  of  hay  fever.  It  is 
wise  to  reinforce  the  pollen  toxin  injections  with  inocu- 
lations of  autogenous  bacterins  prepared  from  the 
complicating  infection.  Such  treatment  has  been  ap- 
plied therapeutically  as  well  as  prophylactically.  It 
is,  however,  of  such  recent  date  that  no  definite  con- 
clusions should  be  drawn.  It  may  share  the  same 
fate  as  Dunbar's  "  pollantin  "  used  to  produce  passive 
immunity  in  pollenosis. 

Diseases  of  Lungs 

Bronchitis. — The  following  bacteria  may  be  identi- 
fied: M,  catarrhalis,  M,  paratetragenus,  B,  influenzcB, 
Streptococcus,  Staphylococcus,  B.  typhosus,  B,  coli,  Z),. 


BACTERIAL  INOCULATION  291 

Pneumonice, Diphtheroid  bacilli,  B,  tuberculosis, Fried- 
lander's  bacillus  and  Streptothrix  actinomycosis.  Pul- 
monary diseases  offer  an  exceptional  field  for  bac- 
terin  therapy,  owing  to  the  unusual  vascularity  of  the 
tissues,  permitting  the  opsonins  to  become  effective. 
Consequently,  brilliant  results  are  achieved  routinely 
both  curatively  and  prophylactically  (Fig.  5Q) ,  This, 
of  course,  does  not  apply  to  tuberculosis  and  actino- 
mycosis nearly  to  the  degree  that  it  does  to  other 
infections. 

Pneumonia, — Essentially  the  same  bacteria  are  to 
be  found  as  in  bronchitis.  Keen  discrimination  must 
be  exercised  not  to  employ  bacterins  if  there  exists  a 
doubt  of  bactersemia,  otherwise  the  employment  of 
autogenous  bacterins  in  adequate  dosage  will  lessen 
mortality  and  shorten  convalescence.  The  opsonic 
index  here  as  in  bronchitis  should  be  utilized  to  secure 
the  best  results.  In  unresolved  pneumonia  and 
bronchopneumonia  of  childhood  a  special  field  of  use- 
fulness is  offered  autogenous  bacterins.  Prophylactic 
immunization  applies  in  pneumonia  the  same  as  in 
bronchitis  and  in  the  event  of  epidemics  should  be 
routinely  practiced.  The  protective  dose  is  1,000,- 
000,000  pneumococci. 

Purulent  Bronchiectasis  and  Pulmonary  Ab- 
scesses, — The  bacteriology  differs  in  no  respect  from 
that  of  bronchitis  and  pneumonia.    The  expectoration 


292  APPLIED  IMMUNOLOGY 

must  be  cultured  and  recultured  frequently  for  the 
preparation  of  the  correct  autogenous  bacterins.  The 
opsonic  index  will  render  service  in  the  proper  manage- 
ment of  the  case.  Although  some  benefit  attends  the 
use  of  bacterins,  the  results  are  not  brilliant,  owing  to 
the  poor  drainage  in  these  chronic  conditions. 

Whooping-cough, — Although  the  bacillus  of 
Bordet-Gengou  is  generally  admitted  to  be  the  cause 
of  pertussis,  early  in  the  attack,  even  before  the  second 
week,  other  bacteria  as  M,  catarrhalis,  B,  influenzce, 
Pneumococcus  and  Streptococcus  complicate  the  in- 
fection. Sufficient  data  have  been  gathered  to  demon- 
strate the  value,  prophylactically  and  therapeutically, 
of  mixed  bacterins  in  this  disease.  Indeed  the  attacks 
have  not  only  been  ameliorated  but  the  period  of  con- 
valescence shortened  fifty  per  cent. 

Diseases  of  Aumentary  System 

Pyorrhoea  Alveolaris  and  Tonsillitis, — The  or- 
ganisms usually  found  include  the  Pneumococcus, 
Streptococcus,  Staphylococcus,  M,  catarrhalis,  spiro- 
chcetce,  vibrios  and  Tubercle  bacillus.  The  results  of 
bacterin  therapy  have  not  been  such  as  to  place  it  as 
a  recommendable  procedure  of  any  great  moment  in 
these  diseases,  unless  supplemented  by  adequate  local 
treatment,  in  the  case  of  pyorrhoea  alveolaris  requir- 
ing the  cooperation  of  a  dentist.  Some  form  of  the 
streptococcus  can  be  cultured  in  at  least  75  per  cent. 


BACTERIAL  INOCULATION  293 

of  cases  and  when  remote  secondary  joint  involve- 
ments arise  and  anaemias  supervene,  autogenous  bac- 
terial inoculations  assisting  effective  oral  treatment 
are  of  great  value.  It  will  be  remembered,  however, 
that  reinfections  are  prone  to  occur,  also  that  unless 
the  patient  is  willing  to  submit  to  dental  cooperation 
and  prolonged  treatment,  inoculations  of  bacterins 
will  prove  of  little  or  no  value.  In  advanced  disease, 
when  the  gums  are  spongy  and  necrosed,  the  teeth 
loose  and  the  alveolar  tissue  in  a  carious  state,  benefit 
may  accrue,  but  cure,  in  its  strict  sense,  is  no  longer 
possible.* 

Acute  tonsillitis  furnishes  no  particular  oppor- 
tunity for  bacterin  therapy.  The  importance  of  the 
tonsils  as  atria  of  infection  in  certain  synovial  arthritic 
and  other  affections  must  not  be  overlooked.  Con- 
sequently in  subacute  and  chronic  disease,  in  peri- 
tonsillitis or  in  aborting  attacks  of  quinsy  autogenous 
bacterins  may  render  signal  service  ( Fig.  45 ) .  Sur- 
gery,  however,  is  indicated  in  many  instances,  in 
preference  to  bacterins. 

Enterocolitis, — Exclusive  of  typhoid  fever,  dys- 
entery and  cholera,  the  bacteria  demonstrable  in  this 
condition,  and  more  particularly  mucous  colitis,  are 
the  colon  group,  the  Streptococcus^  the  Pneumococcus, 

*  Recent  evidence  points  to  Entamoeba  buccalis  as  an  etiological 
factor  in  many  cases  of  pyorrhcea  alveolaris.  In  these  cases  the  local  and 
hypodermic  use  of  emetin  hydrochloride  has  a  markedly  beneficial  effect. 


294  APPLIED  IMIVIUNOLOGY 

the  B,  'pyocyaneus,  the  B,  acidi  lactici  and  rarely  the 
Bacillus  of  Friedldnder.  Bacterin  therapy,  especially 
using  autogenous  suspensions  of  the  first  three  named 
organisms,  has  in  many  cases  been  productive  of  good 
results.  The  initial  dose  had  best  be  smaller  than 
the  average  and  here  the  utilization  of  the  opsonic 
index  as  a  control  of  treatment  will  prove  service- 
able. In  not  a  few  cases  no  material  benefit  has 
followed  the  use  of  bacterins. 

Typhoid  Fever. — Inasmuch  as  the  lesions  of  the 
intestines  stand  out  conspicuously  in  the  pathology 
of  this  disease  and  are  the  organs  first  concerned  by 
the  invading  microbe,  the  B,  typhosus,  it  may  be 
permissible  to  discuss  its  therapy  under  this  heading. 
In  the  first  place,  it  will  be  recalled  that  not  infre- 
quently by  bacteriological  study  the  fever  will  be 
found  to  be  due  to  an  organism  closely  allied  to  the 
bacillus  of  Eberth,  namely  the  B,  paratyphosus  A  or 
B  or  the  paracolon  bacilli,  hence  the  advisability  of 
accurate  bacteriological  investigation  if  this  disease 
is  to  be  treated  biologically.  A  number  of  observers 
have  reported  favorably  as  to  mortality  complications 
and  relapses  by  bacterial  inoculations.  In  spite  of 
the  fact  that  after  the  first  two  or  three  days  a  bac- 
tersemia  supervenes,  enduring  for  a  number  of  days 
or  weeks,  a  strong  movement  is  in  progress  relative 
to    the    routine    employment    of    typho-bacterin    in 


BACTERIAL  INOCULATION  295 

typhoid  fever.  The  argument  is  not  altogether  con- 
vincing, but  the  adoption  of  this  mode  of  therapy  is 
entitled  to  serious  consideration.  In  ten  days  or  two 
weeks,  after  the  bacilli  are  no  longer  demonstrable  in 
the  blood,  the  expert  administration  of  typhoid  bac- 
terin  may  result  advantageously.  It  would  appear 
that  a  stock  bacterin  prepared  from  an  old  culture  of 
high  antigenic  properties  is  preferable  to  an  autog- 
enous preparation.  There  exists  a  danger  of  the  em- 
ployment of  too  large  dosage.  A  primary  inoculation 
of  50,000,000  bacilli  is  proper.  The  opsonic  index 
will  render  service  in  the  control  of  subsequent  in- 
jections (Fig.  53). 

The  bacterin  treatment  of  "  typhoid  carriers," 
whether  the  infection  lurk  in  the  gall-bladder  or 
urinary  tract,  has  at  times  resulted  brilliantly,  at 
others  failed. 

Antityphoid  inoculation  stands  out  as  one  of  the 
particular  bright  lights  in  bacterial  immunization.  In 
the  armies  of  England,  France,  Germany,  Japan, 
and  the  United  States,  adopting  prophylactic  inocu- 
lations, the  fall  in  incidence  and  mortality  rate  from 
typhoid  fever  has  been  remarkable  (Fig.  57).  Were 
the  practice  universal  typhoid  fever  would  soon  cease 
to  exist. 

In  1913,  in  the  army  of  the  United  States,  of 
90,646  inoculated  officers  and  enlisted  men,  only  three 


296 


APPLIED  IMMUNOLOGY 


developed  typhoid  fever  with  no  fatahties.  Anti- 
bodies reach  their  height  usually  within  two  weeks 
after  the  first  inoculation  and  immunity  is  alleged  to 
be  absolute  for  one  or  two  years  and  may  endure  for 
many  more,  although  it  is  advisable  to  reinoculate 
after  one  year  if  an  epidemic  threaten  or  the  individual 
be  exposed.  The  most  approved  doses  for  protective 
inoculation  and  those  adopted  by  the  United  States 


Fig.  57. — Illustrating  typhoid-fever  rates  in  United  States  Army.    Shaded  columns  rep- 
resent decrease  in  incidence  of  typhoid;    black  areas,  the  decrease  in  death-rate. 

Army  are:  First  dose  500,000,000;  second  and  third 
doses  1,000,000,000  each.  Intervals  of  a  week  to  ten 
days  must  separate  the  inoculations.  In  over  500,000 
inoculations  in  the  Army  and  Navy  no  bad  results 
have  been  reported.  Clinically,  the  majority  of  those 
inoculated  develop  fever  rarely  over  101°  F.,  but  at 
times  exceeding  even  103°  F.  Headache,  malaise 
and  muscular  aches  are  commonly  observed;  rarely 
chills,  nausea  and  vomiting  occur.    Agglutinins  and 


BACTERIAL  INOCULATION 


297 


opsonins  are  readily  demonstrable  (Fig.  58).  The 
time  is  ripe  when  it  is  imperative  that  physicians, 
nurses,  ward  attendants  and  all  those  coming  in  con- 
tact with  typhoid  patients  should  be  immunized.    This 


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Fig.  58. — Antityphoid  inoculation  or  immunization.    Observe  the  rapid  rise  in  antibody 
formation  as  demonstrated  by  the  opsonic  index  curve. 

rule  should  include  all  individuals  travelling  in  and 
residents  of  typhoidal  districts  as  well  as  the  members 
of  families  and  pupils  of  schools  in  which  the  disease 
has  made  its  appearance. 


298  APPLIED  IMMUNOLOGY 

Dysentery. — Bacterin  therapy  concerns  only  the 
bacillary  type  of  this  disease.  Closely  allied  bacilli 
have  been  described  by  Kruse,  Shiga,  Flexner,  Hiss 
and  Strong.  Bacterial  inoculations,  employing 
mostly  the  Kruse- Shiga  type  of  organism,  have  been 
extensively  used  in  all  save  the  acute  gangrenous  form 
of  the  disease,  with  the  result  that  the  mortality  has 
fallen  from  6.3  to  0.9  per  cent.  It  should  be  noted 
that  in  acute  cases  bacterin  therapy  is  contra-indicated 
from  the  fourth  to  the  twenty-first  day.  During  this 
period  Kruse  and  Shiga's  anti-endotoxic  serum  should 
be  administered  in  full  doses. 

Cholera. — Bacterin  therapy  in  this  disease  is 
limited  entirely  to  preventive  inoculation;  it  is  value- 
less in  a  cm-ative  capacity.  Since  the  introduction  of 
prophylactic  inoculations  with  the  vibrio  cholerse,  the 
incidence  of  the  disease  in  a  study  of  thousands  of 
uninoculated  and  inoculated  cases  has  fallen  from  3.6 
to  0.66  per  cent.  Three  immunizing  doses  of  500, 1000 
and  1000  million  spirilla  respectively  are  administered 
at  intervals  of  about  ten  days. 

Diseases    of    Cardiovascular,    Lymphatic    and    Nervous 
Systems,  also  Other  Acute  Specific  Fevers 

Bactercemia,  Septicoemia  and  Pycemia, — It  is  our 
belief,  for  reasons  previously  stated  under  "  Contra- 
indications," that  bacterial  inoculations,  in  these  con- 


BACTERIAL  INOCULATION  299 

ditions,  are  not  only  valueless,  but  harmful,  and  should 
the  patient  recover,  he  does  so  not  because  of,  but  in 
spite  of,  bacterin  therapy. 

Endocarditis, — The  streptococcus  of  the  mitior  or 
viridans,  foecalis  or  salivarius  and  pyogenes  longus 
types,  the  pneumococcus,  staphylococcus,  gonococcus 
and  B.  influenzce  are  held  to  be  the  responsible  in- 
fective bacteria.  The  results  of  bacterial  inoculations 
have  been  far  from  promising,  although  a  few  suc- 
cesses have  been  reported  in  the  chronic  and  even  in 
the  subacute  forms  of  the  disease.  Treatment  in  this 
disease,  if  conducted  at  all,  must  be  with  the  utmost 
caution.  In  the  acute  form,  it  is  certainly  condemn- 
able. 

Rheumatic  Fever  and  Articular  Rheumatism, — 
The  Streptococcus  rheumaticus  is  accepted,  in  many 
quarters,  to  be  the  causative  organism.  The  results 
of  Buchanan  in  the  acute  stage  of  these  affections  and 
those  of  Veitch  in  the  chronic  form,  where  entirely 
satisfactory  results  were  obtained  in  50  per  cent,  of 
cases  treated,  are  worthy  of  consideration,  but  as  yet 
have  not  been  duplicated  by  confirmatory  in- 
vestigation. 

Malta  Fever. — The  M,  melitensis  is  the  offending 
bacterium.  Although  the  authors  have  had  no  ex- 
perience, it  is  claimed  on  good  authority  that  small 
doses  in  the  acute  stage  and  larger  dosage  in  the 


SOO  APPLIED  IMMUNOLOGY 

chronic  stage  have  been  followed  by  undoubted  benefit 
in  the  treatment  of  this  disease. 

Cerebrospinal  Meningitis, — It  is  probable  that  the 
value  of  meningo-bacterin  is  to  prevention  as  anti- 
meningitis  serum  is  to  cure.  As  a  result  of  the  im- 
munization of  over  10,000  persons,  none  receiving 
full  immunizing  dosage  developed  epidemic  spinal- 
meningitis.  Albright  states  that  "  meningo-bacterin 
is  of  as  much  value  as  a  prophylactic  in  meningitis  as 
typho-bacterin  is  in  typhoid  fever." 

For  purposes  of  protection,  three  doses  of  500,000,- 
000,  1,000,000,000  and  1,000,000,000  meningococci 
respectively  are  administered  at  intervals  of  a  week  or 
ten  days. 

Hydro  phobia, — Although  the  exact  nature  of  the 
virus  of  rabies  has  not  been  definitely  determined, 
antirabic  inoculation  as  suggested  by  Pasteur  has 
been  extensively  practiced  and  its  value  universally 
recognized.  In  a  curative  capacity  rabies  vaccine 
seems  to  be  ineffective. 

After  immediate  and  thorough  cauterization  of  the 
wound,  inflicted  b}^  an  animal  having  or  suspected  of 
having  rabies,  the  individual  should  waste  no  time  in 
undertaking  the  "  Pasteur  Treatment."  In  the  mean- 
time, the  animal  should  not  be  killed,  but  securely 
confined  and  observed;  if  rabic,  it  will  die  in  a  few 
days  manifesting  symptoms  of  the  disease.     If  the 


BACTERIAL  INOCULATION  301 

animal  has  been  killed,  as  is  commonly  the  case,  the 
head  and  attached  neck  should  be  sent  to  a  State  or 
municipal  laboratory  for  examination  for  the  char- 
acteristic "  negri  bodies." 

Antirabic  inoculation  consists  of  a  series  of  daily 
subcutaneous  injections  of  virus  prepared  by  emulsi- 
fying the  specially  dried  spinal  cord  of  rabbits,  dead  of 
rabies  from  a  fixed  virus.  Rabies  vaccine  is  best  pre- 
pared in  a  laboratory  particularly  devoted  to  that  pur- 
pose. A  number  of  pharmaceutical  firms  and  rabies 
institutes,  to-day,  on  request,  make  daily  deliveries 
in  caloris  bottles  of  the  amount  for  administration, 
making  it  unnecessary  for  the  patient  to  patronize 
an  "  institute  "  and  rendering  it  perfectly  feasible  for 
the  family  physician  to  conduct  the  treatment  at  home. 
All  that  the  practitioner  is  obliged  to  do  is  to  note  the 
age  of  the  patient,  the  date  and  hour  of  the  bite,  its 
location  and  extent,  if  possible  whether  or  not  the 
animal  surely  had  rabies,  and  furnish  these  data  to  the 
expert  or  firm  about  to  produce  the  vaccine.  In  gen- 
eral the  "  Pasteur  Treatment  "  comprises  twenty-five 
inoculations  administered  over  a  period  of  three  weeks. 
On  the  first  day  three  inoculations  are  given  four 
hours  apart,  on  the  second  and  third  days  two  in- 
jections are  made  at  intervals  of  six  hours;  on  the 
fourth  and  succeeding  days  only  one  inoculation  is 
administered. 


302  APPLIED  IMMUNOLOGY 

Allusion,  however,  should  be  made  to  the  more 
recent  work  of  Semple  and  Harris,  who  have  prepared 
efficient  vaccines  from  the  medullas  of  rabbits,  which 
will  keep  for  long  periods  and  are  constantly  ready 
for  use,  thereby  obviating  the  elaborate  methods  of 
Pasteur.  Harris  in  particular  has  reported  on  the  in- 
transpinal  injection  in  animals  of  0.1  milligramme  of 
powder  obtained  from  the  pulverized  frozen  brain  and 
cord  of  animals  killed  by  a  fixed  virus.  This  prepara- 
tion is  dried  in  vacuo  over  sulphuric  acid  at  a  low  tem- 
perature. It  is  claimed  to  produce  immunity  in  a  few 
hours  or  days,  instead  of  weeks,  and  can  be  preserved 
in  sealed  tubes  indefinitely.  Further  investigation  is 
necessary  to  determine  whether  or  not  Harris' 
preparation  is  to  supplant  the  more  familiar  "  Pasteur 
Treatment." 

Malignant  Neoplasmata 

All  attempts  to  produce  an  efficient  tumor  extract 
or  emulsion  for  the  treatment  of  carcinoma  and  sar- 
coma have  proved  futile.  A  few  years  ago  Doyen 
proclaimed  the  specificity  of  the  Micrococcus  neo- 
formans  in  the  etiology  of  cancer.  This  organism  is 
midoubtedly  an  accidental  invader,  and  to  the  best  of 
our  knowledge  no  case  of  carcinoma  has  been  or  will 
be  cured  by  the  agency  of  neoformans  bacterin. 

Coley's  treatment  of  sarcoma  is  worthy  of  mention. 


BACTERIAL  INOCULATION  303 

Coley's  fluid,  so-called,  is  a  mixed  bacterin  of  the 
streptococcus  of  erysipelas  and  the  Bacillus  prodigi- 
osus.  It  is  indicated  (1)  in  all  cases  of  inoperable 
sarcoma,  excepting  the  melanotic  type;  (2)  for  two 
or  three  weeks  even  in  operable  cases,  if  there  be  a 
chance  of  saving  a  limb,  that  is  when  the  giant-celled 
type  of  tumor  exists;  (3)  as  a  prophylactic  against 
recurrence  after  operation,  and  (4)  post-operatively, 
even  against  carcinomatous  recurrence.  Apparently, 
a  large  number  of  properly  selected  cases  have  been 
cured  by  administering  daily  inoculations,  starting 
with  one-quarter  minim  in  the  gluteal  or  pectoral 
region,  and  increasing  the  size  of  the  dose  one-quarter 
minim  daily  until  a  reaction  is  evoked,  that  is  a  tem- 
perature of  102°  to  104°  F.  The  dosage  must  not  be 
increased  and  may  have  to  be  diminished  on  the  super 
vention  of  reaction.  It  is  recommended  that  the  inocu- 
lations be  made  subsequently  in  the  tumor  if  the  pa- 
tient be  not  too  susceptible.  The  initial  tumor  in- 
jection should  be  only  one-quarter  of  the  previous 
inoculation  into  the  gluteal  or  pectoral  region.  Coley 
states  that  carcinoma  is  not  influenced  by  the  treat- 
ment. In  the  treatment  of  430  cases  of  inoperable 
sarcoma  he  reports  47  cases  of  complete  cure,  in  28 
of  which  there  was  no  recurrence  for  three  to  fifteen 
years.    He  asserts  that  as  a  prophylactic  after  opera- 


304  APPLIED  IMMUNOLOGY 

tion  the  use  of  the  bacterial  toxins  has  reduced  the 
percentage  of  recurrence  from  75  to  less  than  25 

per  cent. 

Yeast  and  Sour  Milk 

Brewer's  yeast  {Saccliaromyces  cerevisice)  has 
been  a  popular  remedy  in  the  treatment  of  furuncu- 
losis.  In  many  cases  it  appears  to  be  without  effect 
and  to-day  is  being  supplanted  by  staphylococco-bac- 
terins.  The  employment  of  yeast  in  tuberculosis  and 
cancer  has  been  a  failure. 

MetchnikofF  observed  that  the  peasants  in  certain 
parts  of  Bulgaria,  whose  staple  diet  consisted  in  the 
consumption  of  peculiar  soured  milks,  lived  to  an 
advanced  old  age.  A  peculiar  lactic  acid  bacillus, 
the  Bacillus  lactis  Bulgaricus  or  Bacillus  of  Massol, 
was  shown  to  be  the  effective  organism.  Accordingly, 
tne  ingestion  of  sour  milk  ha§  become  a  world-wide 
practice  in  the  hope  of  avoiding  senility,  correcting 
putrefactive  intestinal  processes  and  a  host  of  other 
affections,  as  infantile  diarrhoea  and  enterocolitis, 
intestinal  indigestion  and  auto-intoxication,  diabetes, 
rheumatism,  gout,  arteriosclerosis,  etc.  Obviously, 
the  results  have  not  measured  up  to  expectations, 
although  benefit  seems  to  have  attended  the  use  of 
artificially  prepared  soured  milk  in  many  cases. 

There  are  many  tablets  on  the  market  for  imme- 
diate ingestion  or  for  making  buttermilk,  purporting 


BACTERIAL  INOCULATION  305 

to  fulfil  MetchnikofF's  assertion,  but  none  of  these 
equal  in  therapeutic  value  the  living  liquid  cultures  of 
the  B.  Bulgaricus.  These  cultures  are  best  prepared 
in  tubes  as  is  done  by  certain  pharmaceutical  houses. 
The  content  of  one  tube  is  poured  into  three  table- 
spoonfuls  of  sweetened  water  and  drunk,  or  it  may  be 
poured  into  a  glass  or  half  a  glass  of  milk  and  drunk 
immediately.  Fresh  cultures  should  always  be  ob- 
tained and  stored  in  a  refrigerator  to  maintain  activity. 
They  may  be  taken  indefinitely  without  harmful 
effect. 


20 


APPENDIX 

PART  A 

SERUM  TREATMENT  OF  HEMORRHAGE 

NORMAL  FRESH  SERUM— PRECIPITATED  HORSE  SERUM- 
TRANSFUSION  OF  BLOOD 

Normal  Fresh  Serum 

The  most  frequent  cause  of  persistent  hemorrhage  from 
small  vessels  is  a  defect  in  the  coagulation  of  the  blood. 
Without  going  into  the  details  of  the  theory  of  coagulation 
of  the  blood,  it  is  now  believed  that  delayed  coagulation  is 
generally  due  to  a  deficiency  of  thrombin  or  fibrin  ferment  in 
the  blood-serum.  In  cases  of  pathological  hemorrhage  this 
deficiency  may  be  supplied  by  the  administration  of  normal 
blood-serum,  and  obstinate  and  persistent  hemorrhage  has 
frequently  been  checked  by  this  means  after  all  other  agencies 
had  failed.  This  is  now  recognized  as  the  best  form  of  treat- 
ment for  persistent  capillary  hemorrhage  whether  inherited 
or  acquired,  such  as  that  due  to  haemophilia,  hemorrhage  of 
the  new-born,  puerperal  hemorrhages,  pulmonary,  intestinal, 
nasal,  and  renal  hemorrhage,  and  traumatic  hemorrhage. 
While  theoretically  human  serum  should  be  more  effective  in 
treatment,  yet  Clowes  and  Busch  (N.  Y,  Med.  Jour.y  Jan.  4, 
1913)  have  shown  that,  practically,  horse  serum  answers  the 
purpose  just  as  well,  and  is  more  available.  In  emergencies, 
when  normal  horse  serum  cannot  be  quickly  obtained,  diph- 
theria antitoxin  may  be  employed  with  good  results. 

Administration  and  Dosage. — The  serum  is  usually  ad- 
ministered hypodermically,  but  may  also  be  given  intraven- 
306 


APPENDIX  307 

ously  or  by  mouth.  The  dose  depends  upon  the  severity  and 
cause  of  the  bleeding,  age  of  the  patient,  etc.  The  average 
adult  dose  is  SO  c.c.  hypodermically  or  10  c.c.  intravenously. 
If  necessary,  a  larger  dose,  up  to  50  c.c,  may  be  given  in  two 
or  three  days,  and  this  may  be  succeeded  by  100  c.c.  a  few 
days  later.  There  is  no  hard  and  fast  rule  as  to  the  size  and 
repetition  of  doses.  One  must  be  guided  entirely  by  the 
effects.  Large  doses  can  be  given  without  injury.  The 
dangers  of  anaphylaxis  may  be  avoided  by  repeating  the 
injections  at  intervals  of  less  than  10  days.  In  children 
it  is  well  to  begin  with  a  dose  of  not  more  than  10  c.c. 

Normal  horse  serum  may  be  applied  locally  for  the 
control  of  hemorrhage  from  wounds.  It  may  be  injected 
through  a  ureteral  catheter  in  cases  of  renal  hemorrhage. 

Precipitated  Sebum 

Clowes  and  Busch  (loc.  cit,)  first  suggested  that  precipi- 
tated horse  serum  be  employed  in  place  of  the  less  convenient 
fresh  serum.  To  quote  from  their  conclusions :  "  Blood 
serum  precipitated  by  means  of  a  suitable  mixture  of  acetone 
and  ether  is  fully  as  effective  as  fresh  serum,  if  not  sup-  x'ior 
to  it.  Precipitated  serum  is  freely  soluble  and  possesses  the 
advantage  over  fresh  serum  of  being  sterile,  always  available, 
and  retaining  indefinitely  its  capacity  to  stimulate  coagu- 
lability of  the  blood." 

Precipitated  horse  serum  may  now  be  obtained  in  the 
form  of  sterile  powder  under  the  name  of  "  Coagulose." 
This  is  easily  dissolved  in  sterile  water,  and  is  then  ready  for 
injection.  The  powder  itself  may  also  be  applied  to  wounds 
and  bleeding  surfaces.  This  powder  retains  its  coagulating 
properties  almost  indefinitely,  while  fresh  serum  loses  its 
effectiveness  in  a  short  time. 


308  APPLIED  IMMUNOLOGY 

Transfusion  of  Blood 

There  are  many  conditions  under  which  transfusion  of 
blood  from  one  individual  to  another  is  of  great  value. 
Among  the  commonest  of  these  are:  Loss  of  blood  follow- 
ing operations  and  accidents,  severe  ansmia  from  various 
causes,  hemorrhage  in  typhoid  fever,  gastric  hemorrhage, 
hemorrhage  of  the  new-born,  and  postpartum  hemorrhage. 
The  introduction  of  new  blood  fulfils  three  functions :  ( 1 )  It 
stops  the  hemorrhage;  (2)  replaces  lost  blood;  (3)  assists 
in  overcoming  any  infection  present  by  supplying  fresh 
complement  and  antibodies. 

In  transfusion  the  person  yielding  the  blood  is  known 
as  the  donor,  wliile  the  person  receiving  it  is  known  as  the 
recipient.  In  selecting  a  donor,  it  is  important  that  his  blood 
be  compatible  with  that  of  the  recipient.  Incompatibility 
of  the  two  bloods  may  be  manifested  in  haemolysis  or  agglu- 
tination of  one  case  by  the  other,  producing  grave  symptoms 
or  even  fatal  results.  Haemolysis  of  the  blood  of  either  the 
donor  or  the  recipient  in  transfusion  is  a  pathological  phe- 
nomenon and  causes  haemogiobinuria  which  may  be  fatal. 
Th*.  'e  is  evidence  that  cases  in  which  haemolysis  occurs  in 
vitro  will  also  show  haemolysis  in  vivo,  and  a  prehminary  test 
of  the  bloods  is  therefore  important  in  the  selection  of  a 
donor. 

It  has  not  been  proved  that  agglutination  of  the  red 
blood-cells  of  the  donor  by  the  serum  of  the  recipient,  or 
vice  versa,  gives  rise  to  serious  results,  and  this  is  therefore 
not  regarded  as  a  contra-indication  to  the  transfusion.  In 
case,  however,  there  should  be  a  considerable  range  of 
selection  of  donors,  one  should  be  selected  if  possible  whose 
blood  is  not  agglutinative. 

In  every  case  where  practicable,  the  blood  of  the  donor 


APPENDIX  309 

should  be  subjected  to  the  Wassermann  test,  to  avoid  the 
introduction  of  syphilitic  infection  into  the  recipient. 

Simple  clinical  tests  for  haemolysis  and  agglutination  have 
been  described  by  Epstein  and  Ottenberg  (Arch.  Int.  Med., 
May,  1909).  They  have  devised  a  technic  in  which  only 
very  small  quantities  of  blood  are  required,  and  which  is 
easy  of  performance.  The  blood  may  be  obtained  by  punct- 
uring the  finger  as  in  the  case  of  the  Wassermann  test.  One 
or  two  c.c.  of  blood  are  allowed  to  drop  into  a  tube  containing 
an  excess  of  1  per  cent,  sodium  citrate  in  normal  salt  solu- 
tion. This  is  centrifuged  to  wash  the  red  cells,  which  can 
be  made  up  to  any  desired  percentage.  Another  cubic 
centimetre  of  the  blood  is  collected,  allowed  to  coagulate, 
and  the  serum  to  separate.  In  studying  haemolysis,  the 
mixture  of  serum  of  the  donor  and  red  cells  of  the  recipient, 
and  vice  versa,  must  be  made  within  12  or  at  most  24  hours 
of  collecting  the  blood.  One  volume  of  corpuscle  suspension 
and  one  or  more  volumes  of  serum  are  drawn  up  into  a 
Wright  capillary  pipette,  thoroughly  mixed,  and  incubated 
in  an  upright  position  for  two  hours.  At  the  end  of  this 
time  the  occurrence  of  haemolysis  can  be  easily  noted  if  it 
has  taken  place. 

It  is  found  that  a  near  blood-relative  (sister  or  brother) 
of  the  patient  is  the  most  satisfactory  donor,  with  the 
smallest  chances  of  untoward  results.  A  robust  person 
should  of  course  be  selected  if  possible. 

Many  methods  have  been  devised  for  the  transmission  of 
blood  from  one  person  to  another.  Some  of  these  are  direct, 
i.e.,  by  the  anastomosis  of  a  vessel  of  the  donor  with  that  of 
the  recipient.  Other  methods  are  indirect,  i.e.,  by  collec- 
tion of  blood  from  the  donor  in  a  suitable  vessel,  and  sub- 
sequent introduction  of  it  into  the  recipient.     Brewer  and 


310  APPLIED  IMMUNOLOGY 

others  have  devised  paraffin-lined  glass  tubes  for  the  passage 
of  blood  from  the  vessel  of  the  donor  to  that  of  the  recipient. 

Crile  was  one  of  the  first  to  employ  the  direct  method 
of  transfusion  extensively,  and  he  unites  the  radial  artery 
of  the  donor  to  a  superficial  vein  in  the  arm  of  the  recipient 
by  means  of  a  special  clamp  {Keen's  Surgery,  Vol.  V,  p.  616). 
Numerous  other  instruments  have  been  recommended  for  this 
purpose,  among  others,  those  of  Landon  {Jour,  A,  M.  A,, 
Aug.  16,  1913)  and  McGrath  {Jour.  A.  M,  A,,  Jan.  3, 
1914). 

The  difficulties  of  blood-vessel  anastomosis  have  led 
many  surgeons  to  devise  various  forms  of  apparatus  for 
removing  blood  from  the  donor,  preventing  it  from  clotting, 
and  subsequently  introducing  it  into  the  vein  of  the  recipient. 
An  advantage  of  indirect  transfusion  is  that  the  amount 
of  blood  utilized  can  be  exactly  measured.  Kimpton  and 
Brown  {Jour,  A,  M,  A.,  July  12,  1913)  have  devised  a 
glass  cylinder  lined  with  paraffin,  into  which  they  withdraw 
the  blood,  which  is  considerably  delayed  in  clotting  by  the 
paraffin. 

Probably  the  simplest  method  of  indirect  transfusion 
is  that  recommended  by  Dorrance  {Penna,  Med,  Jour., 
Sept.,  1914).  The  blood  is  removed  from  a  vein  of  the 
donor  by  means  of  a  50  c.c.  all-glass  syringe,  in  which  has 
been  previously  placed  10  c.c.  of  a  10  per  cent,  solution  of 
sodium  citrate  to  prevent  clotting.  After  withdrawal  of  a 
syringeful  of  blood,  the  syringe  is  detached,  the  needle  re- 
maining in  the  vein  of  the  donor  in  case  more  blood  is  re- 
quired, the  lumen  being  closed  with  a  stilette.  The  blood 
mixed  with  sodium  citrate  solution  is  then  injected  through 
a  needle  similarly  introduced  into  a  vein  of  the  recipient. 
Dorrance  states  that  with  experience  this  operation  can  be 


APPENDIX  311 

performed  so  rapidly  that  no  fluid  to  prevent  coagulation 
need  be  added. 

Two  technical  procedures  can  be  carried  out  to  guide  the 
operator  in  the  quantity  of  blood  to  be  transfused.  One  of 
these  is  the  estimation  of  the  haemoglobin  percentage  of  the 
blood  of  the  recipient  from  time  to  time  during  the  operation, 
and  the  other  is  noting  the  coagulation  time  of  the  blood. 
Many  methods  have  been  employed  for  the  determination  of 
the  coagulation  time,  that  of  Dorrance  {Am.  Jour.  Med.  Sc, 
Oct.,  1913)  being  accurate  and  practical, 


PART  B 

ORGANOTHERAPY 

THYROID  GLAND— ADRENAL  GLAND— PITUITARY   BODY- 
OVARY— CORPUS  LUTEUM— THYMUS  GLAND 

It  has  been  definitely  established  that  the  so-called  ''  duct- 
less glands  "  furnish  secretions  that  have  important  func- 
tions in  the  development  and  metabolism  of  the  body  tissues, 
some  of  them  being  essential  to  life.  In  the  case  of  certain 
of  these  glands,  the  function  of  their  secretion  is  known  with 
some  degree  of  accuracy.  In  the  case  of  others,  our  knowl- 
edge is  indefinite  or  unknown. 

It  is  proposed  here  to  give  brief  mention  to  the  various 
derivatives  of  the  ductless  glands,  with  indications  for  the 
therapeutic  use  of  those  which  have  proved  of  value. 

Thyroid  Gland 

For  many  years  it  has  been  well  known  that  atrophy 
or  removal  of  the  thyroid  gland  causes  marked  disturbances 
of  nutrition,  producing  characteristic  symptoms  grouped 
under  the  names  cretinism  (congenital  absence  of  thyroid 
gland)  and  myxoedema.  This  disturbance  is  believed  to  be 
due  to  defective  tissue  oxidation,  depending  upon  the  absence 
from  the  circulation  of  the  internal  secretion  of  the  gland. 
It  has  been  shown  that  the  thyroid  tissue  contains  more 
iodine  than  any  other  tissue  of  the  body,  and  that  its  activity 
is  directly  proportional  to  the  amount  of  iodine  present. 
This  iodine  is  present  in  the  form  of  iodized  proteid  (iodo- 
thyrin,  thyroprotein,  etc.).  Abnormal  activity  of  the  gland- 
secreting  substance  produces  the  symptoms  found  in  the 
acute  stage  of  exophthalmic  goitre  or  Graves's  disease. 

For  therapeutic  purposes  sheep's  thyroid  is  employed. 
312 


APPENDIX  318 

Extract  of  thyroid  gland  tissue  is  indicated  in  cases  of 
undeveloped  thyroid  (cretinism)  and  in  all  conditions  where 
the  normal  activity  of  the  gland  is  impaired  (myxoedema 
following  atrophy  from  disease  or  loss  of  the  gland  by 
operation).  The  most  brilliant  results  in  the  whole  domain 
of  organotherapy  have  been  seen  in  thyroid  extract  feed- 
ing in  cretinism.  In  these  cases  it  is  customary  to  begin 
with  a  dose  of  5  grains  of  the  crude  extract  three  times  a  day 
in  capsule  form.  This  may  be  gradually  increased  accord- 
ing to  the  effects  produced.  Thyroid  extract  has  also  been 
employed  with  success  in  a  great  number  of  other  conditions 
in  which  defective  oxidation  is  supposed  to  be  a  factor, 
especially  obesity,  certain  skin  diseases,  and  joint  affections. 
It  may  be  used  sometimes  with  good  results  in  the  second 
or  atrophic  stage  of  exophthalmic  goitre,  where  there  is  a 
functional  insufficiency  of  the  thyroid  gland,  but  never  in 
the  acute  or  early  stage.  Numerous  attempts  have  been 
made  to  isolate  the  active  principle  of  the  gland  in  a  more 
or  less  pure  form,  leaving  behind  the  toxic  products.  Beebe 
has  succeeded  in  obtaining  a  thyroprotein  or  concentrated 
extract  of  the  thyroid  gland  which  is  standardized  to  con- 
tain 0.33  per  cent,  of  iodine.  By  the  use  of  this  standardized 
product  the  dosage  can  be  more  accurately  controlled  than 
in  the  case  of  the  crude  extract.  The  principal  symptoms 
that  the  therapeutic  limit  in  thyroid  administration  is  being 
approached  are  rapidity  of  the  pulse,  overactivity  of  the 
sweat  glands,  and  marked  loss  of  weight. 

Adrenal  GiiAND 

The  functional  activity  of  the  adrenal  glands  is  due  to  a 
substance  known  under  various  names,  such  as  adrenalin, 
suprarenin,  epinephrin,  etc.  This  substance  is  a  heart 
stimulant  and  powerful  vasoconstrictor.  For  therapeutic  pur- 


314  APPLIED  IMMUNOLOGY 

poses  it  is  obtained  from  the  ox  and  sheep,  and  is  also  pre- 
pared synthetically.  It  is  questionable  whether  the  sjTithetic 
product  is  superior  to  the  natural  organ  extract.  Adrenalin 
chloride  is  the  preparation  usually  employed,  and  is  generally 
supplied  in  a  1-1000  solution.  It  may  also  be  obtained  in 
powder  or  tablet  form.  Adrenalin,  on  account  of  its  vaso- 
constrictor effects,  is  chiefly  employed  to  control  hemor- 
rhage from  small  vessels,  and  is  useful  as  a  topical  applica- 
tion in  the  treatment  of  various  nasal  conditions,  such  as 
epistaxis,  conditions  requiring  shrinkage  of  the  turbinates, 
hay  fever,  etc.  The  constricting  effects  of  adrenalin  are  very 
transitory,  and  are  usually  followed  by  marked  dilatation. 
Adrenalin  is  used  extensively  as  an  adjuvant  to  cocaine, 
novocaine,  and  other  local  anaesthetics.  The  effects  of  these 
drugs  are  considerably  enhanced  and  prolonged  by  the  vaso- 
constrictor effects  of  adrenahn.  In  local  anaesthetic  mixtures, 
adrenalin  chloride  is  generally  used  in  the  strength  of 
1-10,000.  The  cardiac  stimulant  effects  of  adrenalin  are 
made  use  of  in  the  treatment  of  shock.  In  this  condition 
it  may  be  given  intravenously  in  normal  salt  solution  in  the 
proportion  of  1  drachm  of  adrenalin  chloride  to  a  pint  of 
salt  solution. 

Suprarenal  gland  feeding  has  been  employed  without 
benefit  in  Addison's  disease,  which  is  due  to  atrophy  or 
disease  of  the  adrenals. 

PiTuiTAEY  Body 

Much  knowledge  of  the  pituitary  gland  or  hypophysis 
and  its  secretions  has  been  gained  within  the  past  few  years. 
Experiments  have  shown  that  the  anterior  lobe  of  the  pitui- 
tary body  secretes  a  substance  concerned  in  vital  processes, 
and  which  is  essential  to  life.  The  secretion  of  the  posterior 
lobe  or  pars  nervosa,  while  not  essential  to  life,  contains  a 


APPENDIX  315 

pressor  substance,  which  has  a  marked  action  in  maintaining 
continuously  high  blood-pressure  by  virtue  of  its  production 
of  peripheral  vascular  constriction  and  augmentation  of  the 
force  of  the  heart-beat.  The  effects  of  this  substance  upon 
the  circulation  are  more  powerful  and  lasting  than  those  of 
suprarenal  extract.  Pituitary  extract  in  addition  produces 
diuresis  by  a  specific  action  on  the  renal  epithelium,  and  is 
a  powerful  stimulant  to  involuntary  muscle. 

Therapeutic  Uses. — Extract  of  the  posterior  lobe  of  the 
pituitary  body  is  used  extensively  as  a  stimulant  to  uterine 
contractions  in  the  second  stage  of  labor,  and  is  superior  to 
ergot  and  other  oxytocics.  It  is  very  effective  in  cases  of 
uterine  inertia,  often  rendering  unnecessary  the  use  of  low 
forceps,  and  hastens  the  course  of  labor  in  cases  of  moder- 
ately narrow  pelvis.  It  checks  any  tendency  to  severe  post- 
partum hemorrhage.  The  administration  of  pituitary  ex- 
tract does  not  suffice  to  induce  labor  or  abortion.  The 
dose  of  the  extract  is  1  c.c.  intramuscularly,  and  repeated 
in  one  hour  if  necessary.  In  addition  to  its  uses  in  obstetrics, 
posterior  lobe  extract  is  administered  in  various  conditions. 
It  is  used  by  Cohen  as  a  cardiovascular  support  in  pneumonia. 
He  employs  it  hypodermically  in  doses  of  1  c.c.  every  three 
or  four  hours.  Pituitary  extract  is  said  to  be  useful  in 
preventing  abdominal  distention  due  to  temporary  paralysis 
of  the  bowel  after  abdominal  operations,  in  surgical  shock, 
and  various  other  conditions. 

Contra-indications. — Pituitary  extract  on  account  of  its 
marked  tendency  to  increase  blood-pressure  is  to  be  used  with 
great  caution  in  myocarditis,  arteriosclerosis  and  nephritis. 
In  obstetrical  cases  it  should  not  be  employed  in  abnormally 
narrow  pelvis,  or  in  threatened  rupture  of  the  uterus. 

In  cases  with  symptoms  of  glandular  deficiency,  such  as 
acromegaly,  etc.,  prolonged  feeding  with  powdered  extract 


316  APPLIED  IMMUNOLOGY 

of  the  whole  pituitary  gland  is  indicated,  as  in  these  cases 
probably  both  lobes  of  the  gland  are  functionally  insufficient. 
It  is  stated  that  moderate  doses  can  be  given  for  a  long 
period  without  harmful  effects. 

Ovary 

Ovarian  substance  for  therapeutic  use  is  a  dry  powder 
prepared  from  the  entire  fresh  ovary  of  the  hog.  It  is 
employed  with  some  success  in  functional  dysmenorrhoea, 
disturbances  of  the  menopause,  etc.  In  dysmenorrhoea,  2  to 
4  grains  may  be  given  every  four  hours  until  the  symptoms 
are  relieved. 

COEPUS  LUTEUM 

The  corpus  luteum  of  the  ovary  produces  an  internal 
secretion  whose  function  appears  to  be  to  stimulate  the 
menstrual  flow  and  also  to  sensitize  the  uterine  mucosa  for 
placental  formation. 

Powdered  corpus  luteum  has  been  extensively  used  in 
various  forms  of  dysmenorrhoea,  neurasthenia,  functional 
disorders  associated  with  the  natural  or  artificial  menopause, 
and  functional  amenorrhoea.  It  has  been  found  useful  in 
vomiting  of  pregnancy. 

The  average  dose  of  corpus  luteum  is  five  grains  of  the 
dried  powder  taken  three  times  daily,  during  meals. 

Thymus  Gland 

The  thymus  gland  is  a  mass  of  lymphoid  tissue  situated 
in  the  upper  anterior  part  of  the  thorax.  It  is  well  developed 
at  birth,  and  then  under  normal  conditions  gradually  dis- 
appears. Little  is  known  of  the  function  of  the  thymus,  but 
it  is  believed  to  be  concerned  in  growth  and  development 
and  to  be  closely  associated  in  function  with  the  thyroid. 


APPENDIX  317 

Thymus  gland  has  been  used  empirically  in  a  number  of 
conditions,  among  which  may  be  mentioned  exophthalmic 
goitre,  rickets,  tuberculosis,  haemophilia,  marasmus,  etc., 
with  varying  success.  Calf  thymus  is  supplied  in  desiccated 
form,  and  is  employed  in  doses  of  2  to  4«  grains  three  times 
daily. 

In  addition  to  those  mentioned,  preparations  from  several 
other  organs  of  animals  have  been  used  for  therapeutic  pur- 
poses, such  as  mammary  gland,  testicle,  parathyroid,  parotid 
gland,  spleen,  etc.,  the  results  of  which  have  not  been  of 
sufficient  importance  to  warrant  further  consideration  at  this 
time. 


PARTC 

CHEMOTHERAPY 

ADMINISTRATION  OF  SALVARSAN  AND  NEOSALVARSAN, 
INTRAVENOUSLY,  INTRAMUSCULARLY  AND  INTRA- 
SPINALLY— AUTOSALVARSANIZED  AND  ARTIFICIALLY 
SALVARSANIZED  SERUM 

The  relationship  existing  between  the  Wassermann  reac- 
tion and  the  treatment  of  syphilis  is  so  closely  interwoven 
as  to  warrant  a  chapter  on  the  subject  of  chemotherapy  in 
a  treatise  on  serology.  It  is  not  our  purpose  to  present  a  de- 
tailed consideration  of  chemotherapeutics,  but  simply  to 
refer  to  this  recent  branch  of  scientific  medicine,  particularly 
with  respect  to  the  administration  of  salvarsan  and  neosal- 
varsan. 

It  is  generally  conceded,  all  things  being  equal,  that  im- 
munotherapy, theoretically,  is  superior  and  should  take  pre- 
cedence to  chemotherapy.  The  reason  for  this  is  at  once 
apparent,  due  to  the  fact  that  the  former  is  parasitotropic 
and  not  at  the  same  time  organotropic.  Unfortunately  bi- 
ologic therapeusis  has  not  been  applicable  to  syphilis,  con- 
sequently the  treatment  of  this  disease  devolved  upon  drugs 
or  chemicals,  some  of  which,  in  spite  of  the  time  required  to 
effect  cure,  have  come  to  be  known  as  "  specific,"  for  example 
mercury. 

HisTOEY  OF  Salvarsan  and  Neosalvarsan 

Ehrlich,    attracted   by   the   alleged   value   of    atoxyl   in 
trypanosomiasis,  and  mindful  of  the  possibility  of  increas- 
ing the  antiseptic  effect  of  drugs,  at  the  same  time  diminish- 
318 


APPENDIX  319 

ing  the  toxic,  by  the  introduction  of  the  halogen  group  into 
the  benzine  ring,  conceived  the  idea  that  arsenic  in  certain 
combinations  could  be  so  compounded  that  its  administra- 
tion in  a  sufficiently  large  dose  would  not  be  injurious  or 
toxic  to  the  organism  and  would  at  the  same  time  destroy 
all  spirochsetse,  sterilizing  the  system.  Repeated  trials  en- 
abled Ehrlich  and  his  collaborators  to  produce  a  synthetic 
drug,  No.  606  in  the  experimental  series,  later  styled  "  sal- 
varsan,"  the  use  of  which  was  claimed  to  be  a  "  therapia 
sterilisans  magna  "  for  the  cure  of  syphilis.  Subsequently, 
further  refinement  led  to  the  production  of  neosalvarsan  or 
No.  914,  apparently  less  toxic  but  likewise  less  efficient. 

Be  it  understood  that  the  treatment  of  syphilis  by  arsen- 
ical preparations  is  not  by  any  means  a  new  idea.  Syph- 
ilologists  for  decades  have  recognized  the  benefit  to  patients 
when  arsenic  supplemented  or  alternated  mercury  and  iodine, 
but  owing  to  the  toxicity  of  the  trioxide  of  arsenic,  it  has  been 
necessary  to  administer  it  in  minimal  dosage.  Consequently, 
during  recent  years  other  forms  of  arsenic,  less  toxic  and  of 
higher  drug  content,  have  been  synthesized,  recommended  and 
utilized.  Among  these  arsenical  preparations  may  be  men- 
tioned the  arylarsonates  (soamin  and  orsudan),  atoxyl, 
arsacetin,  arsenophenylglycin  paramidophenylarsenoxide  and 
sodium  cacodylate.  Some  of  these  have  fallen  into  almost 
complete  disrepute  owing  to  gastro-intestinal  disturbances, 
nephritis  and  neurological  manifestations,  including  blind- 
ness, occasioned  by  their  administration,  and  all  have  failed 
to  measure  up  to  the  virtue  of  salvarsan  and  neosalvarsan. 

Valuable  and  important  as  Ehrlich's  discovery  has 
already  proved  itself  to  be  in  the  treatment  of  syphilis,  ad- 
mittedly the  sheet-anchor  in  the  pharmacology  of  that  dis- 
ease, it  is  extremely  doubtful  if  salvarsan  will  prove  to  be 


320  APPLIED  IMMUNOLOGY 

the  universal,  omnipotent,  all-sufficient  panacea  for  lues 
originally  claimed  by  its  advocates,  although  the  authors 
have  repeatedly  observed,  in  the  primary  stage  of  the  disease 
before  the  advent  of  a  positive  Wassermann,  a  single  inten- 
sive dose  of  salvarsan  suffice  to  effect  a  cure,  thereby  ful- 
filling the  dictum  of  Ehrlich.  Possibly  the  cure  not  attained 
by  a  single  dose  may  be  accomplished  by  repeated  injections 
or  a  combination  with  mercury  and  iodine.  This  much  only 
is  assured  at  present,  that  in  the  great  majority  of  patients 
the  immediate  effect  of  salvarsan  properly  administered  is 
a  symptomatic  cure;  concerning  the  remote  results,  addi- 
tional time  must  elapse  before  definite  conclusions  can  be 
drawn. 

Chemico-physical  Properties. — The  Ehrlich-Bertheim- 
Hata  arsenical  preparation  No.  606,  synthetically  known  as 
dioxy-diamido-arseno-benzene-dihydrochloride,  may  be  repre- 
sented by  the  following  structural  formula : 

HO.  ,0H 

^CeHa  —  As  =  As  -  Ceiiz<^ 
HCINh/  ^NHaHCI 

It  is  commercially  known  in  the  market  at  present  as  "  sal- 
varsan," and  appears  as  a  light  yellowish  powder,  markedly 
acid  in  reaction  and  very  unstable  on  exposure  to  the  atmos- 
phere. For  these  reasons  the  drug  must  be  neutralized  and 
prepared  in  solution  strictly  fresh  each  time  before  its  ad- 
ministration. 

Ehrlich  observed  that  formaldehyde-sulphoxylates  of  so- 
dium possess  the  property  of  restraining  for  a  time  the  auto- 
oxidation  of  solutions  of  dioxy-diamido-arseno-benzene,  and 
by  the  interaction  of  the  two  evolved  dioxy-diamido-arseno- 
benzene-monomethane-sulphinate  of  sodium  or  No.  914,  com- 


APPENDIX  321 

mercially  known  as  "  neosalvarsan."    It  may  be  represented 
by  the  following  structural  formula: 

As  As  As 

NH,  I       1  =  11  NH,  +  HO.CHASONa  =  NH,  |       |  = 


As 

I  NaCHjO.SONa-f  HjO 
OH 


( 


Neosalvarsan  appears  as  an  orange-yellow  powder  of  pecu- 
liar odor,  dissolving  very  easily  in  cold  water  with  completely 
neutral  reaction. 

Neosalvarsan  is  even  more  unstable  than  salvarsan,  and 
although  slightly  less  trouble  to  prepare  for  administration, 
requires  even  greater  precaution  as  to  freshness  and  tem- 
perature of  solution,  owing  to  its  greater  proclivity  to  oxidi- 
zation and  the  formation  of  products  of  a  high  degree  of 
toxicity.  Unfortunately,  its  ease  of  solution  in  water,  at 
once  in  a  neutral  state  ready  for  administration,  and  better 
toleration  by  patients,  does  not  outweigh  the  greater  effec- 
tiveness and  spirochaeticidal  power  of  salvarsan,  the  injec- 
tion of  which  with  proper  care  and  technic  is  attended  with  a 
minimum  degree  of  reaction. 

Indications. — Salvarsan  or  neosalvarsan  is  indicated  in 
all  stages  of  syphilis,  and  should  be  the  first  thought  in  treat- 
ment, provided  no  contra-indications  exist.  Indeed,  in  the 
early  primary  stage  cure  may  often  be  obtained  by  the  ad- 
ministration of  salvarsan  alone,  particularly  before  the  ap- 
pearance of  a  positive  Wassermann  reaction.  It  seems  es- 
pecially effective  in  those  cases  where  mercury  and  iodine 
have  failed.  In  cerebrospinal  syphilis  the  effects  are  vari- 
able. For  instance  in  gummatous  meningitis,  whether  diffuse 
21 


322  APPLIED  IMMUNOLOGY 

or  localized,  with  expressions  as  epilepsy,  monoplegia,  etc., 
remarkable  results  are  observed.  In  tabes  dorsalis,  partic- 
ularly if  the  intravenous  administration  of  the  drug  be  sup- 
plemented with  intraspinal  injection  of  autosalvarsanized  or 
artificially  salvarsanized  serum,  there  is  reasonable  hope  of 
arresting  the  disease  if  attacked  in  its  infancy.  In  paresis 
and  encephalitis  the  authors  have  never  observed  noteworthy 
benefit,  the  diseases  exhibiting  continuous  retrogression.  The 
drug  is  of  great  value  in  hereditary  lues  and  in  pregnant 
syphilitics. 

The  provocative  employment  of  salvarsan  and  neosalvar- 
san  is  a  matter  of  considerable  importance.  The  arsenical 
preparations  similar  to  mercury  may  be  utilized  to  provoke 
a  positive  Wassermann  reaction  in  a  syphilitic  patient  other- 
wise exhibiting  a  negative  serological  reaction.  Thus  in 
patients  who  have  previously  received  antisyphilitic  treat- 
ment, or  in  whom  the  clinical  evidence  for  syphilis  is  strong 
and  the  ordinary  Wassermann  reaction  has  resulted  nega- 
tively, the  so-called  provocative  dose  of  salvarsan,  neosalvar- 
san  or  mercury  should  be  employed,  and  the  blood-serum 
again  tested  at  the  end  of  twenty-four  to  forty-eight  hours. 
It  has  been  our  practice  to  use  0.1  gramme  of  salvarsan  or 
0.15  gramme  of  neosalvarsan,  administered  intravenously, 
for  this  purpose.  The  explanations  offered  for  the  employ- 
ment of  arsenical  and  mercurial  preparations  in  this  role  are 
that  the  positive  serological  reaction  is  due  to  the  liberation 
of  endotoxins  from  the  killed  spiroch^etse  or  to  the  stimula- 
tion of  the  latent  spirochastse  by  a  dose  of  the  drug  in- 
sufficient to  effect  a  complete  destruction.  The  phenomenon 
or  biochemical  reaction  is  doubtless  closely  related  to  the 
Herxheimer  reaction. 

In  non- syphilitic  diseases,  salvarsan  enjoys  an  extensive 
employment.      Indeed   in   relapsing   fever,   tertian   malaria, 


APPENDIX  323 

framboesia  (yaws  or  pian),  filariasis  and  Vincent's  angina, 
salvarsan  appears  to  be  equally  as  specific  as  in  syphilis. 
It  may  be  emplo3'ed  in  nervous  disorders  in  which  arsenical 
medication  is  indicated  and  exerts  either  a  beneficial  or  cura- 
tive effect,  according  to  Best,  in  acanthosis  nigricans,  ulcus 
tropicum  or  phagedoenicum,  variola,  verrucae  planae,  Syden- 
ham's chorea,  scurvy,  dermatitis  herpetiformis,  quartan  and 
tropical  malaria.  Thus  in  many  diseases  the  tonic,  stimula- 
tive and  alterative  action  of  arsenic  is  linked  with  the  germi- 
cidal effect  of  the  drug.  Good  or  indifferent  results  have 
been  reported  in  Aleppo  boil  (Oriental  sore),  ansemia,  kera- 
tosis follicularis,  leprosy,  lichen  planus,  lupus  vulgaris,  my- 
cosis fungoides,  pellagra,  pityriasis  rubra,  tuberculosis  and 
experimental  tick  fever. 

In  chancroid,  bilharziasis,  Hodgkin's  disease,  psoriasis, 
scarlet-fever,  trichinosis,  sarcoma,  carcinoma  and  trypano- 
somiasis, the  drug  appears  to  be  without  appreciable  effect. 
In  explanation  of  the  last  it  is  alleged  that  the  trypanosomes 
are  more  susceptible  of  being  rendered  arsenic-fast  than  are 
the  spirochaetse. 

In  veterinary  medicine  salvarsan  has  rendered  signal  ser- 
vice and  a  specific  effect,  particularly  in  pleuropneumonia 
of  horses  and  African  glanders  (lymphangitis  epizootica). 

Acquired  Resistance  of  Spirochcetce  to  Salvarsan. — It  is 
generally  known  that  trypanosomes  possess  the  power  of 
adapting  themselves  to  circumstances,  for  instance  preser- 
vation against  injurious  influences.  In  other  words,  they 
may  become  immune  to  their  own  antibodies  or  chemicals  di- 
rected against  them,  as  arsenical  preparations.  This  resis- 
tive property  then  becomes  a  characteristic  of  the  organism 
and  may  be  transmitted  from  generation  to  generation.  Al- 
though this  characteristic  has  never  been  demonstrated  to 
obtain  for  the  spirochaeta  pallida,  it  is  presumed  from  anal- 


S24  APPLIED  IMMUNOLOGY 

ogy  and  clinical  experience  that  spirochaetae  pallida  may  also 
exhibit  drug-fast  properties.  Thus  are  explained  the  failures 
of  mercury  to  influence  syphilis  after  the  first  few  months  in 
many  cases.  In  like  manner,  the  spirochaetae  may  become 
tolerant  or  resistant  to  the  influence  of  arsenic  when  ad- 
ministered in  salvarsan  and  neosalvarsan.  It  would  appear 
that  early,  small  or  sublethal  doses  of  these  drugs  may  be 
responsible  for  the  production  of  arsenic-fast  or  immune 
spirochaetae.  Consequently,  the  inference  is  strong  that  the 
treatment  of  syphilis  should  be  intensive  from  the  start, 
thereby  avoiding  relapses,  significant  of  immunity  on  the 
part  of  the  spirochaetag.  This  is  best  accomplished  by  repeated 
full-sized  intravenous  injections  of  salvarsan,  the  number 
being  controlled  by  the  stage  of  the  disease  and  the  Wasser- 
mann  reaction. 

Contra-indications  and  Precautions. — Salvarsan  is  con- 
tra-indicated in  advanced  degenerative  diseases  of  the  central 
nervous  system,  in  severe  non-syphilitic  retinal  and  optic 
disease,  in  marked  disturbances  of  the  cardiovascular  sys- 
tem, as  acute  endocarditis,  myocarditis,  with  or  without 
nephritis,  extensive  degeneration  of  the  blood-vessels,  and 
angina  pectoris,  in  any  form  of  non-luetic  nephritis,  in  dia- 
betes, in  aneurism  independent  of  lues,  in  pronounced  foetid 
bronchitis  and  pulmonary  tuberculosis,  and  in  persons  mani- 
festing an  idiosyncrasy  for  arsenic.  Chronic  valvular  heart 
disease,  syphilitic  endarteritis,  aneurism  and  endocarditis 
are  not  contra-indications.  In  cases  of  malnutrition,  ca- 
chexia and  infantile  congenital  syphilis  great  care  as  to 
dosage  must  be  exercised.  In  incipient  tabes,  early  paralysis 
and  epilepsy  of  syphilitic  origin,  salvarsan  can  be  employed 
successfully  only  when  administered  early. 

Certain  precautions  are  imperative  and  must  be  observed 


APPENDIX  325 

for  the  proper,  safe  and  best  administration  of  salvarsan 
and  neosalvarsan.    They  are  as  follows : 

1.  An  exact  technic. 

2.  Observance  of  the  integrity  of  the  hermetically  sealed 
ampoule  and  the  normal  physical  characteristics  of  the  con- 
tained drug. 

3.  Immediate  and  fresh  preparation  of  the  solution  just 
prior  to  administration,  avoiding  improper  degree  of  tem- 
perature. 

4.  Selection  of  the  appropriate  dose  for  the  individual 
case. 

5.  Previous  examination  of  the  patient  to  receive  the  drug, 
particularly  investigation  of  the  renal,  cardiovascular  and 
nervous  systems. 

6.  Assurance  that  any  previous  administration  of  mer- 
cury has  not  caused  kidney  irritation.  Wechselmann,  who  has 
given  over  30,000  doses  of  salvarsan,  cautions  against  the 
combined  use  of  salvarsan  and  heavy  mercurial  treatment, 
and  protests  vigorously  against  employment  of  salvarsan 
after  a  course  of  mercurial  treatment. 

7.  Careful  notation  after  injection  of  a  patient  of  reac- 
tionary phenomena,  with  respect  to  repeated  injections,  both 
as  to  time  and  dose ;  the  most  important  consideration  is  ob- 
viously the  possibility  of  kidney  irritation. 

8.  Caution  as  to  the  resumption  of  vocational  activities 
by  the  patient  too  soon  after  the  administration  of  salvar- 
san and  neosalvarsan. 

Dosage. — Michaelis  has  stated  the  average  dose  of  salvar- 
san to  be  0.01  gramme  to  every  kilogramme  of  body  weight. 
Salvarsan  is  marketed  in  original  packages  of  hermetically 
sealed  ampoules  containing  0.1,  0.2,  0.3,  0.4,  0.5,  0.6,  1.0, 
2.0  and  3.0  grammes ;  of  these  the  average  adult  dose  is 
0.6  gramme,  which  at  times  may  be  advantageously  dimin- 


326  APPLIED  IMMUNOLOGY 

ished  by  0.1  gramme  for  women.  In  children  the  dosage 
should  be  proportionate  to  the  age.  Syphilitic  new-born 
infants  should  receive  from  0.02  to  0.1  gramme  of  the 
drug. 

Neosalvarsan,  although  less  toxic,  is  unfortunately  also 
less  effective  than  its  predecessor,  salvarsan.  The  average 
adult  dose  is  0.9  gramme,  corresponding  in  arsenical  con- 
tent to  0.6  gramme  of  salvarsan.  Commercially  it  is 
marketed  in  ampoules  with  the  following  proportionate 
dosage: 


Dose 

Dose 

0.15  gramme 

neosalvarsan 

=  0.1  gramme 

salvarsan 

0.3 

K 

=  0.2 

te 

0.45 

ii 

=  0.3 

t( 

0.6 

ti 

=  0.4 

" 

0.75 

(C 

=  0.5 

(C 

0.9 

(C 

=z  0.6 

(I 

1.5    grammes 

fC 

=  1.0 

(( 

3.0 

" 

=  2.0  grammes 

(( 

4.5 

(( 

=  3.0 

a 

The  last  three  doses  are  for  veterinary  purposes  only  and 
have  no  place  in  human  therapeutics. 

The  repetition  of  the  dose  is  dependent  upon  the  method 
of  administration  (intravenous  or  intramuscular),  the  or- 
ganic condition  of  the  patient  and  his  susceptibility  to  reac- 
tionary phenomena.  Usually  when  injected  intravenously, 
the  doses  are  repeated  in  five  to  ten  days  in  the  absence 
of  reactionary  contra-indications.  In  some  cases  weekly  ad- 
ministration of  0.6  gramme  of  salvarsan  intravenously  for 
several  weeks  has  been  unproductive  of  harmful  effect.  The 
policy  should  be,  particularly  in  early  syphilis,  to  produce 
an  intensive  or  specific  effect  by  repeated  intravenous  injec- 
tions until  a  negative  Wassermann  reaction  is  obtained.  It 
is  seldom  that  less  than  three  administrations  are  necessary 


APPENDIX  327 

and  often  a  dozen  or  more  injections  are  required  to  render 
the  Wassermann  reaction  negative.  Obviously,  with  feeble 
or  prostrated  patients  of  those  manifesting  a  cardiovascular 
or  central  nervous  s^^stem  lesion,  the  initial  dose,  at  least, 
must  be  smaller  than  the  average. 

When  a  tonic  and  alterative  effect,  in  conjunction  with 
specific  action,  is  desired,  full  doses  of  the  drugs  at  intervals 
of  several  weeks,  may  be  administered  intramuscularly,  or,  if 
merely  the  tonic,  alterative  effect  of  the  drugs  is  desired, 
small  and  frequently  repeated  intramuscular  injections  may 
be  desirable.  An  intramuscular  method  of  administration 
is  that  by  fractional  doses.  0.1  to  0.2  gramme  of  salvarsan 
suspended  in  oil  is  injected  every  other  day  until  a  total 
of  1.2  grammes  has  been  given  to  the  patient.  This  mode 
of  therapy  applies  more  particularly  to  the  treatment  of 
non-syphilitic  diseases.  Small  intramuscular  injections,  lack- 
ing sufficient  remedial  action,  may  invite  relapses,  conse- 
quently it  is  advisable  either  to  precede  or  supplement  the  in- 
tramuscular injection  by  a  full  dose  of  the  drug  intra- 
venousl}^  In  all  stages  of  syphilis,  ^'ith  the  possible  excep- 
tion of  the  early  primary,  it  is  imperative  to  supplement  sal- 
varsan with  mercury  or  mixed  treatment. 

Methods  of  Preparation  and  Administration. — Time  and 
experience  have  evolved  four  methods  -^  for  the  employment 
of  salvarsan  and  neosalvarsan :  the  subcutaneous,  the  intra- 
muscular, the  intravenous  and  the  intraspinal  or  intra-  or 
subdural. 

Following  the  successful  original  experiments  on  animals 
by  Hata,  Hoppe,  in  the  Clinic  of  Professor  Konrad  Alt  in 
Uchtspringe,   on  the   suggestion  of  Ehrlich,  in   September, 

^  The  administration  of  arseno-benzol  by  mouth  is  unworthy  of  con- 
sideration. The  same  objection  obtains  respecting  its  use  as  a  dusting 
powder  for  the  chancre  on  account  of  cost  if  for  no  other  reason. 


S28  APPLIED  IMMUNOLOGY 

1909, was  the  first  to  try  salvarsan  on  the  human  subject.  The 
preparation  used  was  an  alkaline  solution  of  the  disodium 
salt.  Shortly  afterward  Michaelis,  Wechselmann  and  Lange 
devised  the  method  of  neutral  suspension  injections  and  prac- 
ticed the  same  subcutaneously  and  intravenously  in  thou- 
sands of  cases.  Other  notable  methods  that  have  been  ad- 
vised are  Junkemann's  and  Lesser's  modification  of  Alt  and 
Hoppe's  alkaline  method  and  Kromayer's  suspension  of  sal- 
varsan in  paraffin.  About  this  time  Schreiber,  associated 
with  Hoppe,  described  the  method  by  intravenous  administra- 
tion. Wechselmann  appears  to  be  the  first  to  have  employed 
salvarsan  by  intraspinal  injection.  Subsequently,  Marie  and 
Levaditi  likewise  injected  neosalvarsan  directly  into  the 
spinal  canal.  While  the  technic  of  direct  intraspinal  injec- 
tions of  salvarsan  and  neosalvarsan  was  being  perfected,  even 
before  the  use  of  hypertonic  solutions  of  neosalvarsan  as 
practiced  by  Ravaut  and  Wile,  Swift  and  Ellis  recommended 
the  intraspinous  injection  of  auto-salvarsanized  serum.  This 
method  is  less  irritating  to  the  nervous  system  and  less 
dangerous  and  consequently  is  the  most  popular  form  of  sub- 
dural arsenic  medication.  Recently  Fordyce  has  recom- 
mended adding  minute  quantities  of  salvarsan  or  neosalvar- 
san to  the  auto-salvarsanized  serum  prior  to  intraspinal 
injection. 

Subcutaneous  Administration. — The  subcutaneous  admin- 
istration of  salvarsan  or  neosalvarsan,  either  the  injection 
of  the  full  therapeutic  dose  at  once  or  repeated  injections  of 
fractional  doses  over  a  period  of  time,  is  practically  an 
obsolete  method.  This  has  been  due  to  great  and  long  per- 
sistence of  pain,  induration,  non-absorption  of  the  chemical 
and  necrosis  of  the  skin. 

Intramuscular  Administration. — The  intramuscular  in- 
jection is  superior  to  the  subcutaneous  inasmuch  as  the  above 


APPENDIX  329 

noted  objections  are  not  so  marked,  although  not  infre- 
quently encountered,  subsequent  pain  and  induration  being 
the  most  objectionable.  Occasionally  the  indurated  area 
may  undergo  liquefactive  necrosis,  resulting  in  sinus  for- 
mation. 

Owing  to  the  fact  that  all  the  arsenic,  detectable  in  ex- 
creta, is  eliminated  in  three  to  six  days  after  intravenous  ad- 
ministration, while  it  persists  for  a  much  longer  period  fol- 
lowing intramuscular  injection,  it  has  been  recommended  and 
is  practiced  by  some  clinicians  to  supplement  the  intravenous 
in  the  course  of  a  week  by  intramuscular  injections.  The 
rationale  of  this  modus  operandi  is  not  without  reason  and  is 
deserving  of  consideration.  It  can  be  explained  on  the  as- 
sumption that  by  the  intravenous  injection  the  drug  is  at 
once  conveyed  to  the  remotest  parts  of  the  body,  where  it  is 
needed  to  destroy  the  spirochsetse,  thus  producing  a  so-called 
"  intensive "  action  in  contradistinction  to  the  "  perma- 
nent "  action  when  the  drug  is  exhibited  locally  and  an  intra- 
muscular depot  is  established. 

The  technic  of  intramuscular  injections  is  somewhat  vari- 
able. Three  solutions  have  been  extensively  employed:  (1) 
the  alkaline  solution,  (2)  the  oily  suspension  and  (3)  the 
neutral  suspension.  Of  these  the  alkaline  solution  method 
is  to-day  probably  most  commonly  utilized.  The  preparation 
of  the  solution  may  be  similar  to  that  described  under  Intra- 
venous Administration,  differing  only  in  that  for  intramus- 
cular injection  the  drug  is  dissolved  and  injected  in  20  c.c.  of 
fluid,  one-half  the  quantity  being  injected  on  either  side.^  A 
common  procedure  for  the  preparation  of  the  alkaline  solu- 

*  For  the  intramuscular  injection  of  neosalvarsan  a  five  per  cent, 
solution  is  employed,  since  one  gramme  of  the  drug  dissolved  in  twenty- 
two  cubic  centimetres  of  water  makes  an  isotonic  solution.  Therefore, 
for  each  0.15  gramme  of  neosalvarsan  about  three  cubic  centimetres  of 
freshly  distilled  water  should  be  added. 


330  APPLIED  IMMUNOLOGY 

tion  is  to  triturate  0.6  gramme  of  salvarsan  in  a  sterile 
mortar  with  twenty-three  drops  of  fifteen  per  cent,  sodium 
hydroxide  solution,  then  to  dilute  with  distilled  water  to  the 
desired  volume. 

More  recently  intramuscular  injections  have  been  exten- 
sively made  with  simple  suspensions  of  salvarsan  and  neo- 
salvarsan  in  oils,  1 :  10  dilution,  such  as  sesame,  olive 
sweet  almond  and  paraffin.  Certain  pharmaceutical  labora- 
tories market  the  neutral,  water-free  solidified  fat  suspen- 
sions, liquid  at  body  temperature,  in  sterile  ampoules, 
requiring  merely  a  syringe  for  their  administration. 

The  neutral  suspension,  at  one  time  largely  used,  both  in- 
tramuscularly and  subcutaneously,  at  present  enjoys  a  very 
restricted  emplo3"ment.  It  is  prepared  by  triturating  care- 
fully 0.5  or  0.6  gramme  of  salvarsan  with  eight  or  ten  drops 
of  fifteen  per  cent,  caustic  soda  solution  in  a  sterile  porcelain 
dish.  To  this  is  added  at  first  drop  by  drop  with  constant 
trituration  the  required  quantity  of  sterile  water  (5  to 
10  c.c).  The  fine  suspension  thus  produced  is  tested  exact- 
issme  with  litmus  paper  for  neutral  reaction,  and  a  drop 
of  the  soda  solution  or  hydrochloric  acid  added  in  accord- 
ance with  the  reaction. 

The  upper  outer  quadrant  of  the  gluteal  region  is  the 
most  desirable  locality  for  intramuscular  injection,  by 
virtue  of  comparative  freedom  from  nerves  and  blood-vessels 
(Fig.  59).  The  vicinity  of  the  sciatic  nerve  must  be  care- 
fully avoided.  The  injection  should  be  given  deeply  and 
slowly,  thereby  obviating  hemorrhage  and  rupture  of  the  mus- 
cular tissue.  The  skin  is  conveniently  disinfected  at  the  site  of 
injection  by  three  to  five  per  cent,  tincture  of  iodine.  After 
injection  the  fluid  is  distributed  as  widely  as  possible  by 
careful  massage,  and  the  needle  puncture  sealed  with  col- 
lodion.    In  sensitive  patients  the  area  to  be  injected  may  be 


Fig.  59. — Site  for  deep  intramuscular  injection.  The  needle  is  plunged  inward  and 
downward  at  a  point  selected  anywhere  on  the  line  C  Z>,  which  indi<-ate.s  a  region  in  the 
upper  outer  quadrant  of  the  buttock  free  of  large  ve.ssels  and  nerves.  The  line  C  D  les 
two  finger  breadths  below  the  iliac  crest;  the  point  C  located  on  the  vertical  line  -4  tf 
erected  midway  between  the  tuberosity  of  the  ischium  and  the  great  trochanter  of  the 
femur. 


APPENDIX  331 

ana?sthetized,  preliminaril}^,  by  injecting  through  the  needle, 
in  situ,  two  cubic  centimetres  of  a  one  per  cent,  or  five  cubic 
centimetres  of  a  one-half  per  cent,  novocain  solution.  Post- 
ing ectional  pains  or  reactive  painful  infiltrations  may  be 
combated  locally  with  hydrothcrapeutic  measures,  as  the  hot- 
water  bottle,  hot  compresses,  etc.,  and  internally  the  adminis- 
tration of  pyramidon  has  proved  very  effectual.  Patients  had 
best  remain  in  bed  for  some  time  after  the  injection. 

Unfortunately,  no  patient  who  has  ever  received  salvar- 
san  or  neosalvarsan  intramuscularly  desires  it  a  second  time 
by  that  method,  and  any  patient  who  has  had  the  drug  ad- 
ministered both  intravenously  and  intramuscularly  invariably 
prefers  the  former  because  of  the  lesser  pain.  Certainly 
there  are  occasions  when,  by  force  of  circumstances,  it  be- 
comes necessary  or  expedient  to  use  the  intramuscular 
method,  but  these  exceptions  must  be  few  in  number. 

Intravenous  Administration. — The  intravenous  adminis- 
tration of  salvarsan  and  neosalvarsan  in  dilute  solution  is 
the  best  method  and  the  mode  of  therapy  of  the  future.  The 
consensus  of  opinion  among  those  of  widest  experience  is 
that  salvarsan  is  more  effective  than  neosalvarsan.  A  rou- 
tine procedure  with  the  authors  is  to  commence  treatment 
with  neosalvarsan,  which,  if  well  tolerated,  is  supplemented 
by  salvarsan.  We  firmly  believe  in  the  intensive  form  of 
treatment,  with  proper  observance  of  precautions  and  con- 
tra-indications,  attempting  thereby  to  eradicate  the  disease ; 
particularly  is  this  true  in  the  primary  stage  of  syphilis, 
when  the  indication  for  the  intravenous  administration  of 
arseno-benzol  is  just  as  acute  as  the  scalpel  in  a  well-defined 
case  of  appendicitis.  In  the  early  days  of  the  chancre,  es- 
pecially before  a  positive  Wassermann  is  obtainable,  it  is 
possible  to  cure  syphilis  by  one  or  more  injections  of  sal- 
varsan alone;  in  the  late  primary  usually,  and  throughout 


332  APPLIED  IMMUNOLOGY 

the  secondary,  latent,  tertiary,  nervous  and  hereditary  forms 
of  the  disease,  mercury  or  mixed  treatment  must  supplement 
the  administration  of  arseno-benzol.  We  seldom  administer 
less  than  three  intravenous  injections,  usually  at  intervals  of 
a  week.  Then,  after  a  respite  of  three  weeks,  the  blood  is 
tested  by  the  Wassermann  reaction.  If  the  result  still  be 
positive,  three  more  injections  are  given  and  so  on  until  in 
a  few  cases  a  dozen  or  more  doses  have  been  administered. 
As  soon  as  a  negative  Wassermann  is  obtained  the  patient  is 
subjected  to  a  vigorous  course  of  mercurial  or  mixed  treat- 
ment for  three  months  to  a  year.  Treatment  is  then  sus- 
pended and  the  further  conduct  of  the  case  may  depend  upon 
Wassermann  reactions  of  the  blood  taken  at  three-month 
intervals,  associated  in  certain  cases  with  the  Wassermann 
reaction  and  cytological  examination  of  the  spinal  fluid. 
Although  no  absolute  law  may  be  promulgated,  no  case 
should  be  pronounced  cured  until  consecutive  negative  Was- 
sermann tests  are  obtained  for  a  period  of  at  least  two  years 
after  the  suspension  of  all  treatment.  (See  technic  de- 
scribed in  Chapter  XII.) 

Preparation  of  Patient. — After  determining  the  organic 
fitness  of  the  candidate,  the  patient  to  receive  an  intravenous 
injection  of  salvarsan  or  neosalvarsan  should  receive  a 
cathartic  the  night  before  the  injection  of  the  drug.  He 
should  take  no  food  at  the  meal  immediately  preceding  the 
introduction  of  the  remedy,  but  may  be  allowed  limited  quan- 
tities of  liquid.  He  should  be  placed  in  the  supine  position 
on  a  table,  the  arm  (preferably  the  left  in  right-handed  in- 
dividuals) slightly  abducted,  the  forearm  supinated  and 
resting  on  a  support,  the  elbow  very  slightly  flexed,  as 
shown  in  Fig.  60.  While  an  assistant  sterilizes  the  region 
of  the  elbow,  with  soap  and  water,  alcohol  and  a  solution  of 
bichloride,  the  operator,  always  with  the  strictest  observance 


Fig.  60— Position  of  patient  for  intravenous  injection  of  salvarsan.  Note  the 
easily  applied  and  released  rubber  dam  tourniquet  shown  in  the  corner  sketch;  also  the 
manner  ot  fixing  the  skin  overlymg  the  vein  with  the  thumb  as  the  needle  is  introduced 
into  the  vein. 


■'I^.^' 


r 


^ 


M 


>^ 


^     -^ 


-5  "3 
a  to 
°  c 

=3  „ 


APPENDIX  333 

of  the  chemical  and  aseptic  precautions,  prepares  the  solution 
for  intravenous  administration. 

Preparation  of  Salvarsan  Solution. — In  Fig.  61  are 
shown  the  various  reagents,  solutions,  glass  vessels  and  ap- 
paratus useful  for  the  preparation  and  administration  of 
salvarsan  intravenously.  All  apparatus,  as  described  in  the 
outfit  (Fig.  62),  here  recommended,  including  thermometers, 
burette,  etc.,  may  be  sterilized  by  boiling.  The  ampoule  of 
the  drug  is  best  antisepticized  by  submersion  in  a  cold  solu- 
tion of  bichloride  or  formalin,  after  noting  that  it  is  intact 
and  its  content  in  no  way  oxidized.  The  water  and  saline 
solution  employed  for  solution  and  dilution  of  the  salvarsan 
must  be  sterile  and  freshly  distilled.  Chemically  pure  sodium 
chloride  must  be  used  in  making  the  saline  solution.  The 
common  laboratory  or  commercial  distilled  water  is  usually 
not  sterile.  Clinical  experience  has  demonstrated  that  reac- 
tions following  the  administration  of  salvarsan  are  more 
common  when  spring,  tap  or  stale  water  is  used  than  when 
freshly  distilled  water  is  the  diluent.  Whether  or  not  this 
increased  toxicity  is  due  to  the  interaction  of  the  endotoxins 
of  the  contained  flora  and  arseno-benzol  or  is  referable  to 
other  causes,  it  is  advisable  to  employ  freshly  distilled  water. 
This  may  be  readily  obtained  in  sufficient  quantities  by  an 
apparatus  (Fig.  63)  capable  of  being  installed  in  the  physi- 
cian's office.  Sterilization  may  then  be  assured  or  completed 
in  an  Arnold  sterilizer  for  one-half  hour.  The  use  of  sterile 
freshly  distilled  water  and  chemically  pure  salt  solution  and 
the  preparation  of  the  salvarsan  solution  immediately  before 
intravenous  injection  are  imperative  conditions. 

Twenty  to  forty  cubic  centimetres  of  warm  (110°  to 
120°  F.)  sterile  freshly  distilled  water,  (W,  Fig.  61)  are 
placed  in  the  graduated  glass  stoppered  cylinder  or  mixer 
(M)   containing  a  dozen  or  more  small   glass  balls.     The 


334  APPLIED  IMMUNOLOGY 

neck  of  the  ampoule  (A)  containing  salvarsan  (usually  0.6 
gramme)  is  nicked  with  the  small  file  accompanying  the 
commercial  package  and  the  neck  easily  broken.  The  con- 
tent is  emptied  into  the  mixing  cylinder  and  the  substance  en- 
tirely dissolved  by  shaking,  producing  a  clear  light-yellowish 
solution  of  a  strongly  acid  reaction.  In  order  to  fit  this 
solution  for  human  administration  it  must  be  neutralized. 
Accordingly  1.14?  cubic  centimetres  or  approximately  2S 
minims  of  a  freshly  prepared  fifteen  per  cent,  solution  of 
purified  sodium  hydroxide  (C)  are  added.  For  this  purpose 
a  medicine  dropper  (D)  is  convenient.  The  following  table, 
dependent  upon  the  quantity  of  salvarsan  utilized,  may  be  of 
service : 

Salvarsan  16  per  cent.  Solution  of  Sodium  Hydroxide 

0.6  gramme  requires  1.14  c.c.  or  approximately  23  to  24  minims. 


0.5 

0.95    "      " 

19 

'  20 

0.4 

0.76    "      " 

15 

'  16 

0.3 

0.57    "      " 

12 

0.2 

0.38    "      " 

8 

0.1 

0.19    "      " 

4 

The  addition  of  the  caustic  soda,  which  should  be  in  one 
quantity  and  not  added  slowly  drop  by  drop,  produces  a 
heavy  whitish  yellow  precipitate,  redissolved  in  excess  on 
shaking;  if  necessary  another  drop  or  two  of  the  caustic  soda 
solution  may  be  added.  At  this  juncture  the  solution  should 
be  neutral  and  perfectly  clear,  resuming  its  original  light 
yellowish  coloration.  It  is  advisable  now  to  add  just  one  more 
drop  of  the  soda  solution,  which  will  render  the  solution  very 
faintly  alkaline.  Slight  turbidity  or  cloudiness  of  the  solu- 
tion is  an  indication  that  insufficient  sodium  hydroxide  has 
been  added.  If  in  doubt  the  reaction  should  be  tested  with 
litmus  paper  (L)  and  the  neutrality,  weak  alkalinity  or 
acidity  of  the  solution  definitely  determined.  If  decidedly 
alkaline,  a  drop  or  two  of  the  dilute  hydrochloric  acid  (H) 


Fig.  64. — Illustrating  method  of  eliminating  air  from  tubing.  The  burette  and 
needle  are  alternately  elevated  and  lowered,  and  w.hen  the  tubing  is  entirely  free  of  ail 
air  bubbles,  the  two  stop-cocks,  one  in  the  needle  and  the  other  in  the  burette,  are  turned 
off,  leaving  the  tubing  and  needle  filled  with  normal  saline  solution. 


Fig.  65. — Thomas'  salvarsaii  and  neosalvarsan  burette.  The  length  of  the  rubber 
tubing  between  the  needle  and  transfusion  thermometer  should  not  be  more  than  eight 
inches,  and  that  between  the  thermometer  and  burette  proportionately  increased  in 
length. 


APPENDIX  335 

must  be  added.  The  additions  of  caustic  soda  or  hydro- 
chloric acid  can  best  be  controlled  by  taking  them  with 
pipettes  or  droppers  (D)  from  the  medicine  glasses  (G). 
The  rubber  tubing  incorporating  the  transfusion  thermom- 
eter (T)  and  fitted  with  the  platinum-iridium  needle  (P)  or 
the  blunt  infusion  needle  (N),  rarely  necessary,  is  adjusted  to 
the  burette,  which  with  the  tubing  is  thoroughly  rinsed  out 
and  the  tubing  and  needle  filled  with  warm  normal  salt  solution 
(S),  the  tubing  being  freed  of  air  by  alternately  elevating 
and  lowering  the  burette  and  needle,  as  shown  in  Fig.  64. 
The  weakly  alkaline  solution  of  salvarsan  is  then  filtered 
through  sterile  cotton  contained  in  the  funnel  (F)  into 
the  graduated  burette  (Fig.  65).  Sufficient  warm  (110° 
to  120°  F.)  sterile  0.5  per  cent,  saline  solution  made  from 
freshly  distilled  water  is  then  passed  through  the  cotton 
filter,  filling  the  burette  to  the  300  cubic  centimetre  mark.^ 
This  is  the  proper  dilution  for  0.6  gramme  salvarsan.  The 
quantity  is  diminished  by  50  cubic  centimetres  for  each  0.1 
gramme  less  of  salvarsan. 

With  the  patient  supine  and  the  arm  moderately  abducted 
and  forearm  supinated,  the  region  of  the  elbow  is  antisepti- 
cized  with  soap  and  water,  alcohol  and  bichloride  or  a  three 

*  In  view  of  the  recommendation  that  the  preferable  diluting  fluid 
for  salvarsan  is  0.5  per  cent,  saline,  while  for  neosalvarsan  it  is  0.4  per 
cent,  salt  solution,  the  authors  have  found  it  practicable  and  con- 
venient from  the  standpoint  of  preparation  of  solutions  to  utilize  two 
standard  solutions — 0.9  per  cent,  chemically  pure  sodium  chloride  solu- 
tion and  freshly  distilled  water.  From  these  any  desired  strength  saline 
solution  can  be  readily  prepared.  For  instance,  in  diluting  the  salvarsan 
solution,  distilled  water  is  added  until  the  quantity  in  the  burette  reaches 
135  cubic  centimetres,  then  0.9  per  cent,  salt  solution  is  added  until  the 
amount  equals  300  cubic  centimetres.  In  the  case  of  the  full-sized  dose 
of  neosalvarsan,  the  dilution  is  made  first  with  distilled  water  to  the 
85  cubic  centimetre  level,  then  0.9  per  cent,  salt  solution  is  added  imtil 
the  quantity  in  the  burette  stands  at  150  cubic  centimetres. 


336  APPLIED  IMMUNOLOGY 

per  cent,  tincture  of  iodine,  and  a  tourniquet  of  rubber  dam 
(R)  is  placed  around  the  arm,  sufficiently  taut  to  render 
the  superficial  veins  about  the  elbow  prominent,  care  being 
exercised  not  to  obliterate  the  radial  pulse  (Fig.  60).  The 
median  cephalic  or  basilic  vein  is  preferable  to  a  vein  exactly 
in  the  cubital  fossa,  because,  should  inflammation  in  or 
about  the  vein  supervene,  the  patient  will  experience  less 
discomfort  on  flexion  and  extension  of  the  elbow.  The  bu- 
rette containing  the  required  quantity  of  salvarsan  solution 
is  given  to  an  assistant  to  hold  or  it  is  suspended  on  a  stand 
adapted  to  the  purpose  (Fig.  60).  At  this  point  it  is  usually 
possible  to  plunge  the  sharp  pointed  platino-iridium  obliquely 
through  the  skin  into  the  vein,  holding  the  needle  with  the 
eye  directed  downward,  at  the  same  time  fixing  the  skin  in 
the  axis  of  the  vein  about  two  inches  distant  from  the  site 
of  puncture  with  the  index  finger  or  thumb  of  the  hand  not 
holding  the  needle.  As  the  needle  is  slowly  and  steadily  ad- 
vanced at  an  angle  of  about  thirty  degrees,  always  keeping  it 
at  the  plane  of  the  axis  of  the  vein,  it  will  usually  be  possible  to 
see  a  slight  dimpling  of  the  skin  overlying  the  vein  at  the 
point  where  the  needle  will  pierce  the  vein.  Just  as  the  sen- 
sation, imparted  to  the  fingers,  of  decreased  resistance  is 
experienced,  as  the  tip  of  the  needle  enters  the  lumen  of  the 
vein,  the  skin  dimple  likewise  disappears.  Rarely,  it  will  be 
necessary  in  the  case  of  young  individuals,  some  women  and 
those  exhibiting  a  thick  layer  of  panniculus  adiposis,  when 
the  veins  are  extremely  small  or  obscured  by  fat,  to  infiltrate 
the  skin  with  a  few  drops  of  a  one  per  cent,  novocain  solution, 
and  then  make  a  cutaneous  incision  one-half  to  three-quarters 
of  an  inch  in  length,  exposing  the  vein.  The  vein  may  or 
may  not  be  lifted  from  its  sheath,  but  in  any  event  it  is  then 
possible  to  introduce  the  needle  without  difficulty,  using 
preferably  the  sharp-pointed  one  or  occasionally  the  well- 


APPENDIX  337 

known  dull-tipped  intravenous  variety  (N,  Fig.  61).  If  it  be 
necessary  to  cut  the  skin,  the  wound  must  be  closed  by  a 
suture. 

Great  care  should  be  exercised  to  see  that  the  tip  of  the 
needle  lies  within  the  vein,  because  the  escape  of  the  salvar- 
san  solution  into  the  subcutaneous  and  perivascular  tissues 
is  productive  of  great  pain,  cellular  infiltration  and  pro- 
longed induration  and  nullifies  the  advantage  of  the  intra- 
venous over  the  intramuscular  method  of  administration. 
For  this  reason,  it  is  well  to  have  the  needle  and  rubber 
tubing  filled  with  normal  salt  solution,  which  will  not  produce 
irritation  should  it  be  allowed  to  escape  into  the  subcutaneous 
areolar  tissue.  If,  by  accident,  a  quantity  of  salvarsan  solu- 
tion should  be  allowed  to  infiltrate  the  perivascular  tissues, 
it  is  advisable  to  incise  the  overlying  skin  immediately,  using 
an  anaesthetic  of  novocain  or  eucaine,  wash  out  the  salvarsan 
with  normal  saline,  and  then  to  puncture  the  vein  directly. 
So  soon  as  the  vein  has  been  entered,  the  tourniquet  is 
loosened,  the  cocks  of  the  burette  and  needle  turned  on, 
and  the  solution  of  salvarsan  permitted  to  flow  very  slowly 
from  the  burette.  The  temperature  of  the  solution  just 
before  entering  the  vein  can  be  read  from  the  thermometer 
and  should  be  blood  heat.  The  introduction  of  the  solution 
should  not  take  less  than  10  minutes.  Just  before  the  sal- 
varsan solution  in  the  burette  falls  to  the  level  of  the  stop- 
cock, it  should  be  turned  off,  cr  the  rubber  tubing  tempo- 
rarily compressed  and  about  twenty-five  cubic  centimetres 
more  of  salt  solution  filtered  into  the  burette,  when  the  com- 
pression is  released  or  the  stop-cock  again  turned  on.  This 
procedure  permits  of  the  patient  receiving  the  full  amount 
of  salvarsan  and  at  the  same  time  suffices  to  wash  the  vein 
free  of  salvarsan,  thereby  avoiding  the  possibility  of  phle- 
22 


S38  APPLIED  IMMUNOLOGY 

bitis,  etc.  A  dry  sterile  gauze  dressing  following  the  removal 
of  the  needle  completes  the  operation. 

A  too  rapid  introduction  of  the  solution  naturally  might 
cause  dilatation  of  the  right  heart  with  its  attendant  re- 
sults. Too  much  care  cannot  be  exercised  to  exclude  air 
bubbles  from  the  apparatus,  although  the  danger  from  air 
embolism  is  not  great — incomparable  in  comparison  with  the 
advent  of  air  through  the  great  veins  of  the  neck,  where  a 
negative  pressure  exists — it  is  nevertheless  a  serious  concern. 
Similarly,  the  filtration  of  all  solution  entering  the  vein  is  a 
matter  of  importance,  lest  solid  particles  of  matter,  as 
glass,  acting  as  emboli,  might  exert  a  harmful  effect.  Throm- 
bosis is  an  accident  alleged  to  have  occurred.  This  must 
supervene  only  as  a  result  of  faulty  technic  or  in  the  presence 
of  an  infection  and  emphasizes  the  necessity  of  thorough 
sterilization  of  all  apparatus  and  material  employed.  It 
is  assuredly  more  likely  to  ensue  when  the  vein  is  exposed, 
due  to  atmospheric  influence  on  the  blood  and  to  metabolic 
changes  in  the  vein  wall.  In  an  experience  covering  several 
thousand  administrations,  it  is  a  complication  which  we  have 
never  encountered. 

Preparation  of  Neosalvarsan  Solution. — The  solution 
of  neosalvarsan  is  slightly  less  trouble  to  prepare  than 
salvarsan,  since  when  dissolved  it  is  at  once  neutral  and 
needs  no  addition  of  alkali  to  counteract  the  acidity.  It  is 
far  more  unstable  than  salvarsan,  consequently  must  be 
used  immediately  after  preparation.  Moreover,  the  solution 
must  never  be  heated.  In  fact,  it  is  emphatically  stipulated 
that  the  temperature  of  the  freshly  distilled  water  or  salt 
solution  must  be  that  of  the  room,  that  is,  68°  to  7^°  F. 
This  had  best  be  determined  by  an  appropriate  thermometer 
(T,  Fig.  61).     Undue  shaking  is  also  to  be  avoided. 

The  content  of  the  full  adult  dose,  0.9  gramme,  is  dissolved 


APPENDIX  339 

in  15  to  20  cubic  centimetres  of  sterile  freshly  distilled  water. 
This  may  be  injected  intravenously,  using  an  appropriate 
syringe,  or  preferably  it  is  filtered  through  cotton  into  the 
burette  and  diluted  up  to  150  cubic  centimetres,  using  a  0.4 
per  cent,  saline  solution  or  freshly  distilled  water.  In  the 
case  of  neosalvarsan,  since  the  quantity  of  solution  is  only 
one-half  that  of  salvarsan,  it  is  allowed  to  enter  the  vein  in 
five  minutes.  In  ail  other  respects  the  technic  of  administra- 
tion differs  in  no  way  from  that  described  for  salvarsan. 

Intraspinal  Administration. — It  was  soon  recognized 
after  the  advent  of  salvarsan  that  its  administration  intra- 
muscularly or  intravenously  in  certain  forms  of  syphilitic 
disease  of  the  nervous  system  was  not  so  effective  as  in 
other  manifestations  of  the  disease.  This  observation  was 
confirmed  by  the  Wassermann  reaction,  inasmuch  as  the 
reaction  of  the  spinal  fluid  in  certain  neurological  cases  per- 
sisted positive  long  after  the  reaction  of  the  blood-serum 
became  negative.  Moreover,  it  is  a  common  observation  to 
find  in  S3'philitic  neurological  patients  a  negative  blood  and 
a  positive  spinal  fluid  Wassermann  or  a  weakly  positive  blood 
reaction  and  a  medium  or  strongly  positive  spinal  fluid  result. 

Communication  between  the  lateral  ventricles  of  the  brain 
and  the  subarachnoid  spaces  of  the  brain  and  spinal  cord 
exists  through  the  foramina  of  Magendie  and  Luschka. 
The  nervous  choroid  plexuses  and  the  velum  interpositum, 
possibly  assisted  by  the  blood-vessels  of  the  meninges,  act 
as  the  chief  dividing  plane  or  filtration  plant  between  the 
blood  and  the  spinal  fluid.  Owing  to  the  fact  that  the 
specific  gravity  of  the  blood  is  greater  than  that  of  the 
cerebrospinal  fluid,  also  that  the  pressure  of  the  cerebro- 
spinal fluid  is  greater  than  that  of  the  intracranial  venous 
blood,  osmosis  takes  place  in  the  direction  of  the  venous 
blood.     This  fact  may  account  for  the  failure  of  salvarsan, 


340  APPLIED  IMMUNOLOGY 

administered  intravenously,  to  reach  the  spinal  fluid  in  any 
considerable  quantity.  In  any  event,  so  far  as  the  effective 
treatment  of  cerebrospinal  lues,  especially  paresis  and 
notably  tabes  dorsalis,  is  concerned,  it  prompted  a  number  of 
investigators  to  apply  both  salvarsan  and  neosalvarsan  di- 
rectly to  the  subarachnoid  spaces  by  spinal  puncture. 
Wechselmann,^  Marinesco,^  Marie  and  Levaditi  ^  were  prob- 
ably the  first  to  employ  this  method.  The  practice  was 
attended  frequently  with  such  marked  reactions,  and  even 
fatalities,  that  it  has  already  become  obsolete.  The  modi- 
fications as  suggested  by  Ravaut"  and  Wile  ^  using  hyper- 
tonic solutions  of  neosalvarsan  are  likewise  not  beyond  re- 
proach and  must  be  regarded  as  dangerous  procedures,  not 
to  be  recommended  for  the  average  case. 

Autosalvarsanized  Serum, — Swift  and  Ellis  ^  have  per- 
fected and  recommended  the  intraspinal  injection  of  the 
patient's  own  salvarsanized  serum,  irrespective  of  the  fact 
that  it  is  impossible  by  the  Marsh  test  to  demonstrate  the 
presence  of  arsenic  in  the  serum  of  patients  shortly  after  the 
intravenous  injection  of  an  adult  dose  of  salvarsan,  where- 
fore if  present  it  must  be  in  minute  quantity,  rendering  the 
modus  operandi  of  the  apparently  beneficial  effect  of  this 
method  of  treatment  difficult  of  comprehension.  Possibly  the 
antibodies  in  the  serum  itself  may  be  a  most  important 
factor.  It  is  unquestionably  true  that  autosalvarsanized 
serum  is  much  less  irritating  and  dangerous  than  direct  sub- 
dural injections  of  salvarsan  or  even  hypertonic  solutions 
of  neosalvarsan.      Consequently,  this  method  is  deservedly 

*Deutsch.  med.  Wochenschr.,  1912,  38,  1446. 

=  Zeitschr.  fiir  Phys.  und  Therap.,  1913,  17,  194. 

« Bull,  et  Soc.  med.  d.  hop.,  Paris,  November  18,  1913. 

^  Ann.  de  Med.,  1914,  1,  49. 

"Jour.  Am.  Med.  Assn.,  1914,  Ixii,  1165;  ibid.,  1914,  Ixiii,  13T. 

» New  York  Med.  Jour.,  July  13,  1912,  53. 


■^^ 


i. 


h 


I 


\  A, 


Fig.  6G. — Showing  position  of  patient  for  spinal  puncture.  The  skin  has  been 
antisepticized  with  iodine  tinr-ture  and  the  needle  introduced  through  the  first  inter- 
vertebral space  above  the  level  of  the  iliac  crests. 


/^ 


/ 


f*  rt 


^  \ 


r 


Fig.  67.— Lumbar  puncture  with  Strauss  needle,  illustrating  the  method  of  intra- 
spinal injection  by  the  gravity  method.  After  introduction  of  needle,  the  obturator, 
O,  is  withdrawn  to  point  shown  in  cut.  This  permits  of  the  spinal  fluid  to  rise  in 
attached  pipette,  P,  graduated  in  hundredths  of  a  cubic  centimetre,  thereby  permitting 
of  the  determination  of  the  degree  of  intraspinal  pressure.  The  desired  amount  of  spinal 
fluid  may  then  be  collected  in  a  test-tube  held  under  the  exit,  M,  by  removing  the 
metallic  plug.  The  plug  is  then  reinserted,  the  pressure  noted  and  the  obturator 
partially  replaced.  The  graduated  pipette  or  tube,  P.  is  then  disconnected  from  the 
needle  at  C  and  the  rubber  tubing,  T",  leading  from  the  re.?ervoir  containing  the  serum 
attached  to  the  needle.  The  obturator  is  again  withdrawn  as  far  as  it  will  go  and  the 
serum  allowed  to  flow  in.  An  assistant  with  his  arm  around  the  patient's  neck,  as  illus- 
trated, and  his  fist  in  the  epigastric  region  can  do  much  to  steady  the  patient  and  assist 
him  to  properly  arch  his  spine. 


^\ 


't 


A 


APPENDIX  341 

the  most  popular  and  best  form  of  intraspinal  treatment  for 
syphilis  thus  far  proposed. 

The  technic,  slightly  modified  from  the  original  of  Swift 
and  Ellis,  is  as  follows:  The  patient  should  receive  the  same 
preliminary  preparation  as  for  salvarsan  or  neosalvarsan,  of 
which  the  former  is  preferable  and  more  effective.  Usually 
the  full  adult  dose  of  salvarsan  is  administered  intravenously. 
Prior  to  the  injection  of  salvarsan  it  is  assumed  that  suffi- 
cient spinal  fluid  has  been  obtained  by  lumbar  puncture 
(Fig.  66)  for  the  various  tests  necessary  in  the  control  of 
the  number  of  intraspinal  injections,  namely,  the  Wasser- 
mann  reaction,  the  lymphocytic  count,  Noguchi's  butyric 
acid  test  for  globulin  or  Nonne's  phase  reaction.  One-half  to 
one  hour  after  the  intravenous  injection  of  salvarsan,  about 
40  cubic  centimetres  of  blood  are  taken  in  centrifuge  tubes 
from  a  vein  of  the  opposite  arm.  This  may  be  allowed  to 
stand  until  the  following  day  for  the  separation  of  the  serum 
or,  as  is  the  practice  of  the  authors,  immediately  centrifuged 
and  IS  cubic  centimetres  of  the  serum  pipetted  off  and  di- 
luted with  18  cubic  centimetres  of  sterile  normal  salt  solution. 
This  forty  per  cent,  dilution  of  serum  is  activated  by  heat- 
ing at  56°  C.  in  a  water-bath  for  one-half  hour.  On  the 
occasions  of  the  second  and  third  treatments,  the  strength 
of  the  serum  is  commonly  increased  to  50  and  60  per  cent, 
dilutions.  The  30  cubic  centimetres  of  diluted  serum  are  then 
carefully  and  slowly  injected  by  lumbar  puncture  after  ap- 
proximately an  equal  amount  of  spinal  fluid  has  been  with- 
drawn. Theoretically,  the  intraspinal  injection  is  best  done 
by  the  gravity  method  (Fig.  67)  ;  practically,  it  can  be  and 
is  properly  accomplished  by  the  faultless  use  of  a  syringe, 
injecting  very  slowly  (Fig.  68).  The  patient  may  be  per- 
mitted to  lie  on  his  side  or  sit  with  his  back  arched.  Im- 
mediately after  the  injection,  the  patient  must  lie  down  and 


342  APPLIED  IMMUNOLOGY 

the  foot  of  the  bed  be  elevated  14  to  16  inches  for  four  hours. 
The  patient  should  continue  in  bed  for  two  or  three  days. 
Repetition  of  the  treatment  depends  upon  the  extent  and 
duration  of  any  reactions  incurred.  As  a  rule  the  intra- 
spinal injections  are  repeated  at  intervals  of  two  weeks.  The 
actual  number  of  treatments  should  depend  upon  the  analysis 
of  the  spinal  fluid,  the  object  being  to  obtain  a  negative  Was- 
sermann  reaction,  a  reduction  in  the  number  of  lymphocytes 
to  five  per  cubic  millimetre,  and  a  normal  globulin  content. 

Artificially  Salvarsanized  Serum, — Attempts  have  been 
made  to  salvarsanize  serum  in  vitro,  in  the  hope  of  increas- 
ing the  arsenical  content  in  view  of  intraspinal  injections. 
The  results  have  been  disappointing  and  in  the  employment 
of  neosalvarsan,  notably  in  Los  Angeles,  disastrous. 

Recently  Fordyce,^*^  adopting  the  technic  of  Ogihde,  in 
which  a  known  amount  of  salvarsan  is  added  to  human  serum, 
recommends  the  following  technic : 

"  Fifty  c.c.  of  blood  are  drawn  into  a  centrifuge  bottle 
and  centrifuged  twice.  It  is  important  to  have  the  serum 
clear  and  free  from  fibrin  and  blood-cells.  To  obtain  the 
requisite  amount  of  the  drug,  old  salvarsan  is  mixed  in  the 
usual  way  in  the  proportion  of  0.1  gm.  to  40  c.c.  of  fluid, 
care  being  taken  not  to  over-alkalinize ;  0.4  c.c.  of  this  solu- 
tion is  the  equivalent  of  1  mg.,  and  is  taken  as  the  standard 
for  measuring  the  dosage.  For  this  purpose  a  1  c.c.  pipette 
graduated  in  hundredths  should  be  employed.  The  desired 
amount  of  salvarsan  is  added  to  from  12  to  15  c.c.  of  the 
serum,  shaken  to  and  fro  to  mix  thoroughly,  and  then  placed 
in  the  incubator  at  37  C.  (98.6  F.)  for  one  hour,  after  which 
it  is  inactivated  for  half  an  hour  at  56  C.  (132.8  F.).  The 
latter  is  a  most  important  step  in  the  technic,  as  Swift  and 

^^Jour.  Am.  Med.  Assn.,  Aug.  15,  1914,  555. 


APPENDIX  343 

Ellis  demonstrated  that  the  splrochaeticidal  properties  of  the 
serum  were  markedly  increased  by  heating. 

"  Salvarsanized  serum  prepared  according  to  the  method 
of  Ogilvie  must  be  used  fresh,  that  is,  within  three  hours  of 
the  time  that  it  is  made  up." 

Although  a  few  investigators  have  added  as  much  as 
12  mg.  of  neosalvarsan  to  the  serum  in  vitro  prior  to  intra- 
spinal injection,  with  salvarsan  an  initial  dose  of  0.1  to 
0.25  mg.,  increased  perhaps  to  0.5  mg., should  not  be  exceeded. 
Pordyce  regards  the  limit  of  safety  to  lie  within  0.5  mg. 

After-care  of  the  Patient. — Following  a  full  intravenous 
injection  of  salvarsan  or  neosalvarsan,  particularly  the 
former,  the  patient  should  lie  still  for  one-half  hour.  Indeed, 
it  were  better  for  him  to  be  confined  to  bed  over  night,  and 
longer  if  reactions  make  it  desirable.  The  practice  of  per- 
mitting patients  to  go  home  alone  immediately  after  an  in- 
travenous injection  of  the  drug  is  reprehensible,  although 
in  many  clinics  and  offices  it  is  commonly  done,  especially 
after  neosalvarsan.  It  is  our  practice  both  in  the  hospital 
and  in  the  office  to  detain  the  patient  for  four  to  six  hours, 
when  if  he  exhibits  no  reaction,  or  any  toxic  effects  of  the 
drug  have  disappeared,  he  is  allowed  to  go  home.  If  the 
reaction  is  persistent  in  any  way,  the  patient  is  advised  to 
remain  over  night. 

After  an  injection  of  arseno-benzol,  the  patient  is  not 
allowed  to  have  anything  by  mouth,  even  water,  for  two 
hours.  If,  then,  he  is  not  nauseated  he  may  be  given  all  the 
water  he  cares  to  drink.  Food  is  usually  not  craved,  but 
should  the  patient  become  hungry  during  the  afternoon  or 
evening  a  glass  of  milk,  broth  or  consomme  with  a  slice  of 
bread  or  a  cracker  is  all-sufficient.  As  a  routine  practice,  it 
is  unnecessary  to  treat  headache,  nausea,  vomiting,  fever, 
diarrhoea,     etc.,     which     occasionally     supervene.       Rarely 


344  APPLIED  IMMUNOLOGY 

pyramidon  for  headache,  a  mustard  plaster  to  the  epigas- 
trium for  gastric  disturbance,  bismuth  subnitrate  for  diar- 
rhcEa  and  an  ice-cap  for  fever  may  be  of  some  value  or  at 
least  pacify  the  patient.  Most  important  is  it  that  the 
urine  be  examined  the  following  day,  in  order  to  learn  the 
extent  of  renal  irritation,  if  present.  If  albumen  or  casts  are 
found,  urinalyses  should  be  performed  daily  to  determine  the 
duration  of  their  persistence,  in  view  of  subsequent  adminis- 
tration of  the  drug.  If  the  evidences  of  kidney  irritation  are 
marked  or  prolonged  for  more  than  24  to  48  hours,  the 
repetition  of  the  injection  becomes  a  serious  consideration 
and  as  a  rule  is  inadvisable.  Arsenical  intoxication  calls  for 
sweat  baths,  either  electric  or  hydrothermic.  Caution  should 
be  enjoined  that  the  patient  not  exert  himself  severely  in  view 
of  cardiac  strain  for  a  few  days,  particularly  in  the  advent 
of  severe  reactions. 

Clinical  Reactionary  Effects  of  Salvarsan. — As  a  rule 
clinical  reactions  are  commoner  and  severer  after  salvarsan 
than  neosalvarsan.  The  primary  dose,  whether  the  former 
or  the  latter,  is  usually  attended  with  greater  reactions  than 
subsequent  injections,  although  the  converse  of  this  is  fre- 
quently observed  owing  to  the  cumulative  effect  of  closely 
repeated  administrations.  The  reactions  probably  occur  by 
virtue  of  two  facts :  Firstly,  the  endotoxins  arising  from  the 
destruction  of  myriad  numbers  of  spirochsetae,  and,  secondly, 
the  toxic  effect  of  arsenic  itself,  based  upon  personal  idio- 
syncrasies. 

During  the  administration  of  salvarsan,  the  patient  not 
infrequently  experiences  a  sensation  of  warmth,  fulness  or 
throbbing  in  the  head,  occasionally  complaining  of  aching 
of  the  teeth  and  a  metallic  taste  in  the  mouth;  his  face  is 
usually  more  or  less  flushed.  If  reactions  supervene,  they 
are  likely  to  be  ushered  in  by  chilliness  or  a  definite  chill 


APPENDIX  345 

about  an  hour  and  a  half  or  two  hours  after  the  injection 
of  the  drug.  This  is  followed  by  headache,  nausea  and  a 
slight  rise  in  temperature  to  99-99 Mi  degrees  F.  Vomit- 
ing may  occur,  after  which  headache  and  nausea  may  dis- 
appear. In  severer  types  of  reaction,  nausea  and  vomiting 
may  persist  and  recur  for  several  hours,  associated  with  a 
temperature  rise  as  high  as  104  degrees  F.  Diarrhoea  is  a 
common  occurrence.  Rarely  a  cutaneous  eruption  of  an 
erythematous  type  due  to  arsenic  may  be  observed.  This 
is  distinctive  from  the  Jarisch-Herxheimer  phenomenon,  since 
it  may  occur  in  non-syphilitic  cases.  Loss  of  appetite  for 
a  few  hours  or  even  a  day  or  two  may  be  observed,  and  occa- 
sionally irritation  of  the  kidneys,  manifested  by  a  cloud 
of  albumen  and  a  shower  of  hyaline  casts,  may  occur.  This 
last  is  a  most  important  warning  prohibiting  in  a  few  in- 
stances the  repetition  of  the  intravenous  injection  of  the 
drug.  Careful  studies  have  never  revealed  injury  to  a 
normal  heart,  kidneys  or  eyes.  It  is  decidedly  reprehensible 
to  administer  salvarsan  to  a  patient  evidencing  renal  irrita- 
tion from  mercurial  treatment.  Wechselmann  has  ascribed 
a  number  of  fatalities  to  this  procedure  and  insists  that  sal- 
varsan should  never  immediately  follow  a  vigorous  course 
of  treatment  by  mercury. 

In  the  vast  majority  of  patients,  receiving  either  salvar- 
san or  neosalvarsan,  we  have  observed  no  sign  or  symptom 
other  than  a  trivial  transient  rise  of  temperature.  Usually 
all  symptoms  disappear  in  six  to  twelve  hours.  As  a  result 
of  treatment,  in  two  or  three  weeks,  the  patient  invariably 
feels  better  physically  and  mentally;  his  ansemia  improves; 
he  gains  in  weight ;  all  lesions  rapidly  disappear  and  the  pa- 
tient is  inclined  to  construe  the  symptomatic  into  a  per- 
manent cure. 

The  Jarisch-Herffheimer  Reaction. — This   phenomenon, 


346  APPLIED  IMMUNOLOGY 

originally  associated  only  with  a  characteristic  cutaneous 
eruption  following  antisyphilitic  treatment,  at  one  time — 
before  the  discovery  of  the  treponema  pallidum  and  the  Was- 
sermann  reaction — enjoyed  considerable  importance  as  a 
diagnostic  aid.  The  skin  and  mucosa  reactions  are  mani- 
fested by  oedema,  swelling,  redness,  pain  and  other  inflam- 
matory signs.  To-day  the  Herxheimer  reaction  is  regarded 
to  be  much  broader  in  its  scope  and  may  be  defined  as  a  cu- 
taneous eruption,  an  aggravation  of  a  pre-existent  syphilitic 
roseola  or  any  inflammatory  reaction  in  syphilitic  tissue  pro- 
voked hy  the  administration  of  salvarsan,  neosalvarsan  or 
mercury.  This  is  to  be  distinguished  from  the  arsenic  rash  in 
non-syphilitics  receiving  salvarsan.  Various  manifestations 
of  the  reaction  are  apparent.  The  increased  redness  of  the 
roseola,  the  inflammatory  reaction  in  mucous  patches,  the 
swelling,  exudation  and  occasional  ulceration  of  gummata 
observed  a  few  hours  or  a  day  or  so  after  medication,  the 
lancinating  pains  of  tabes  dorsalis,  augmented  or  relighted, 
as  observed  following  the  Swift-Ellis  treatment,  the  so-called 
neuro-recurrences  and  the  provocative  positive  Wassermann 
reaction  are  all  evidences  of  the  Herxheimer  reaction  in  whole 
or  in  part. 

According  to  Ehrlich  the  occurrence  of  these  phenomena 
is  due  to  treatment  dosage  insufficient  to  destroy  completely 
the  treponemata  pallida  whereby  the  escaped  viable  or- 
ganisms are  sensitized  or  stimulated  to  increased  activity. 

Excretion  of  Salvarsan  from  the  Body. — The  elimination 
of  salvarsan  from  the  system  has  been  carefully  studied  both 
on  the  human  and  on  lower  animals.  It  has  been  determined 
by  urinalysis  and  gastric  analysis  that  the  elimination  of 
the  drug  occurs  almost  immediately  following  its  intravenous 
administration,  while  after  intramuscular  injection  it  is  sel- 
dom detected  for  an  hour,  although  after  subcutaneous  in- 


APPENDIX  347 

jection  it  has  been  observed  as  early  as  twenty-five  minutes. 
Fischer  and  Hoppe  claim  that  after  intravenous  injections  no 
arsenic  is  demonstrable  after  two  to  three  days,  while  after 
subcutaneous  injections  the  time  limit  is  four  to  five  days; 
after  intramuscular  injections  the  urine  may  show  the  pres- 
ence of  arsenic  for  six  to  ten  days.  Consequently  it  is  the 
natural  supposition  and  the  case  that  tlie  duration  of  elimina- 
tion following  intramuscular  and  subcutaneous  administra- 
tion is  longer  than  after  the  intravenous  injection.  It  is 
alleged  that,  after  the  last  method  of  therapy,  arsenic  has 
been  found  in  the  blood  at  the  end  of  two  days,  and  absent 
at  fourteen  days ;  in  the  urine  for  two  or  three  days ;  in  the 
stools  for  five  or  six  days.  By  the  Marsh  test  the  authors  have 
been  unable  repeatedly  to  demonstrate  the  presence  of  ar- 
senic in  the  blood-serum  thirty,  forty-five  and  sixty  minutes 
after  the  intravenous  injection  of  salvarsan  or  neosalvarsan, 
nor  have  they  succeeded  in  demonstrating  traces  of  the  drug 
in  the  spinal  fluid  one  and  a  half  to  two  hours  following  intra- 
venous administration.  It  is  possible  that  the  mould  test  of 
Gosio,  the  most  delicate  qualitative  test  for  arsenic  that  we 
have,  may  throw  new  light  on  this  subject.  The  test  depends 
upon  the  fact  that  a  certain  mould  {Penicillmm  hrevicaule) 
when  cultured  on  a  medium  containing  arsenic  produces  a 
distinct  odor  of  garlic.  Examination  of  human  milk  at  the 
end  of  three  and  twenty-four  hours  demonstrated  the  absence 
of  the  drug  in  that  secretion.  From  animal  experiments  it  is 
proper  to  infer  that  the  drug  is  entirely  eliminated  from  the 
liver  and  bone  marrow  in  ten  to  sixteen  days.  Further  ex- 
periments show  that  mercury  delays  the  excretion  of  arsenic 
and  explains  why  and  how  mercury  ably  supplements  and 
supports  salvarsan  and  neosalvarsan  in  the  treatment  of 
syphilis.  Potassium  iodide,  on  the  other  hand,  accelerates 
the  elimination  of  arsenic. 


348  APPLIED  IMMUNOLOGY 

Results  of  Specific  Treatment  in  Syphilis, — We  have  four 
ways  of  measuring  the  effect  of  salvarsan  in  the  treatment  of 
syphilis:  (1)  the  disappearance  of  treponemata  pallida,  (2) 
the  disappearance  of  lesions,  (3)  the  recurrence  of  lesions  and 
(4)  the  Wassermann  reaction.  Of  these,  the  first,  constitut- 
ing the  clinical  symptomatology,  is  the  most  important.  How- 
ever, the  value  of  the  Wassermann  reaction,  correctly  and 
competently  performed,  in  the  diagnosis  and  control  of  the 
treatment  of  syphilis,  must  not  be  underestimated,  since,  by 
its  use,  the  diagnosis  in  doubtful  cases  may  be  determined 
and  by  its  periodic  utilization,  recurrence  of  lesions  may  be 
avoided. 

The  earlier  and  more  intensive  the  antisyphilitic  treat- 
ment, whether  by  salvarsan  or  mercury,  the  sooner  and 
more  lasting  are  negative  results  to  be  expected.  A  single 
negative  result  serologically  does  not  signify  cure  and  is 
commonly  observed  in  the  treatment  of  early  syphilis;  the 
Wassermann  reaction  may  rapidly  return  to  positive  if  the 
amount  of  treatment  has  been  insufficient.  On  the  other 
hand,  the  duration  of  negative  reactions  is  directly  propor- 
tional to  the  thoroughness  of  treatment.  Mcintosh  and 
Fildes  state  that  positive  reactions  in  the  secondary  stage  of 
syphilis  may  be  expected  to  become  negative  after  a  single 
course  of  mercury  in  66  per  cent,  of  cases ;  in  tertiary  syph- 
ilis in  33  per  cent,  of  cases.  One-half  to  two-thirds  of  patients 
have  negative  reactions  after  ten  courses  of  mercury  or  two 
years  of  pills.  It  is  safe  to  conclude  that  one-third  to  one- 
half  of  all  patients  treated  by  the  old-fashioned  method, 
although  exhibiting  no  signs  or  symptoms,  judged  from  the 
serological  standpoint  are  or  were  not  cured  of  syphilis.  May 
this  large  percentage  not  include  many  of  those  patients  who 
later  in  life  develop  tabes  dorsalis  and  paresis.'' 

What  has  salvarsan  added  to  the  curability  of  syphilis? 


APPENDIX  349 

Mcintosh  and  Fildes  state  after  the  intramuscular  and  sub- 
cutaneous administration  of  salvarsan  (0.3  to  1.2  gm.)  in 
primary  cases  all  showed  negative  Wassermann  reactions  on 
an  average  of  five  weeks ;  in  the  secondary  stage  many  be- 
came negative  on  an  average  of  eight  weeks;  in  the  early 
latent  form  the  average  was  ten  weeks ;  in  the  tertiary  stage 
little  or  no  effect  was  observed  so  far  as  negative  reactions 
were  concerned.  After  the  combination  of  intramuscular 
with  intravenous  injections  (0.8  to  1.7  gm.),  however,  a 
much  more  pronounced  beneficial  effect  was  observed.  In 
secondary  syphilis  no  case  failed  to  become  negative  and 
did  so  with  great  regularity  in  six  and  a  half  weeks.  In  the 
tertiary  stage  of  the  disease  the  intravenous  method  again 
proved  its  superiority  over  the  intramuscular  and  was  pro- 
ductive of  a  high  percentage  of  negative  reactions.  The 
dictum  of  Ehrlich,  that  in  the  use  of  salvarsan  there  exists  an 
agent  capable  of  curing  syphilis  by  a  single  dose — a  "  thera- 
pia  sterilisans  magna  " — is  true.  Our  experience  has  shown 
that  if  the  diagnosis  of  the  chancre  be  made  sufficiently  early, 
using  the  dark  field  ultra-microscope,  before  the  Wasser- 
mann reaction  becomes  positive,  the  disease  may  be  cured  by 
a  single  dose  of  salvarsan.  The  hope  inspired  by  that  dic- 
tum, however,  has  not  been  fully  realized,  in  view  of  the  fact 
that  only  a  small  percentage  of  cases,  untreated  with  mer- 
cury, yield  a  permanently  negative  Wassermann  reaction 
even  after  repeated  injections  of  salvarsan.  It  is  the  con- 
sensus of  opinion  to-day  that,  as  a  general  rule,  the  most 
appropriate,  efficient  and  speedy  cure  of  syphilis  is  the 
association  of  salvarsan,  preferably  intravenously,  with  mer- 
cury or  mixed  treatment. 


GLOSSARY 

Active  Immunization. — The  process  by  which  the  body  cells 
of  an  animal  are  stimulated  by  a  toxin  or  foreign  body 
(antigen)  to  the  production  of  antibodies  specific 
against  the  given  foreign  substance. 

Agglutination. — A  phenomenon  characterized  by  clumping 
and  loss  of  motility  of  bacteria,  brought  about  by  ag- 
glutinins. 

Agglutinins, — Antibodies  of  the  second  order  of  Ehrlich, 
producing  agglutination. 

Allergy. — The  altered  condition  of  an  animal  into  whose 
tissues  has  been  introduced  an  antigen  or  foreign  cell 
product. 

Amboceptor. — Specific  antibody  of  the  third  order  of  Ehr- 
lich, which  acts  only  in  conjunction  with  non-specific 
substance  or  complement. 

Anaphylaxis. — A  series  of  apparently  deleterious  effects  pro- 
duced by  a  second  injection  of  specific  protein  material 
into  an  animal  that  has  been  previously  "  sensitized  " 
by  a  prior  injection  of  the  same  material. 

Antigen. — ^A  foreign  substance,  usually  of  protein  nature, 
capable  of  exciting  the  formation  of  specific  antibodies. 

Antiserum. — Serum  containing  specific  substances  whereby 
the  action  of  bacteria  or  their  toxins  is  antagonized. 

Antitoxin. — Specific  substance  produced  in  the  blood-serum 
whereby  the  action  of  bacterial  toxins  is  antagonized. 

Autogenous  Bacterin. — Therapeutic  suspension  of  bacteria 
prepared  from  the  particular  strain  cultured  from  the 
infected  patient. 

Autolysis. — Disintegration  of  bacteria  by  treating  them  with 
salt  solution,  alcohol,  ether,  chloroform,  etc.,  theoret- 
ically to  remove  toxic  or  antiopsonic  substances. 
350 


GLOSSARY  351 

Bacterin. — A  suspension  of  bacteria  prepared  for  therapeu- 
tic purposes. 

Bacteriocidin. — Substance  in  the  blood-serum,  capable  of 
destroying  bacteria. 

Bacteriolysin. — Specific  antibody  of  third  order  of  Ehrlich, 
concerned  in  the  dissolution  of  bacteria. 

Bordet-Gengou  Phenomenon. — Complement-fixation  reaction 
as  first  applied  to  infection  with  cholera  spirilla. 

Complement. — Non-specific  substance  normally  present  in 
all  blood-serum,  acting  in  conjunction  with  antibodies 
of  third  order  of  Ehrlich. 

Complement-fixation. — The  using  up,  and  rendering  unavail- 
able for  further  use,  of  complement,  in  the  reaction  be- 
tween antigens  and  their  specific  antibodies  (ambo- 
ceptors). 

Cytolysin. — Specific  antibody  of  third  order,  capable  of  dis- 
solving foreign  cells. 

Endotoxin. — Toxin  bound  up  in  the  bacterial  protoplasm, 
and  only  set  free  by  disintegration  of  the  bacteria. 

Hcemolysin. — Specific  antibody  of  the  third  order  of  Ehrlich, 
capable  of  disintegrating  foreign  red  blood-cells. 

Hcemolysis. — Disintegration  of  red  blood-cells,  setting  free 
the  haemoglobin. 

Hoemolytic  Amboceptor. — Synonymous  with  hgemolysin. 

Immunity. — The  resistance  manifested  by  man  and  various 
animal  species  to  infectious  microorganisms  or  other 
foreign  proteins. 

Immunization. — The  process  by  which  the  state  of  immunity 
is  attained.    It  comprises  two  forms,  active  and  passive. 

Lysins. — Specific  antibodies  of  third  order  of  Ehrlich,  com- 
prising cytolysins,  haemolysins,  bacteriolysins,  etc. 

Opsonic  Index, — Measure  of  the  ratio  of  the  phagocytic 
activity  of  neutral  washed  leucocytes  in  the  patient's 


352  GLOSSARY 

serum  for  given  bacteria,  as  compared  with  those  in  a 
normal  or  control  serum. 

Opsonins. — Specific  substances  in  the  blood-serum  possessed 
of  the  ability  to  sensitize  or  prepare  bacteria  for  phago- 
cytosis. 

Organotherapy. — Therapeutic  administration  of  products 
of  ductless  glands  and  other  organs. 

Passive  Immunization. — The  process  by  which  immunity  is 
acquired  when  artificial  antisera  are  injected  into  the 
animal  body. 

Phagocytosis. — Property  of  the  leucocytes  whereby  they  take 
up  into  their  substance  foreign  particles,  such  as  bac- 
teria, pigment,  carbon  granules,  etc.,  thus  removing 
them  from  the  circulation. 

Precipitins. — Antibodies  of  the  second  order  of  Ehrlich, 
formed  in  the  blood  in  response  to  unorganized  protein 
material. 

Sero-hacterin. — Bacterins  prepared  by  treating  bacteria 
with  their  specific  immune  serum,  thus  "  sensitizing " 
them  so  that  they  are  acted  upon  by  the  complement  in 
the  patient's  blood  immediately  after  injection. 

Stock  Bacterin. — A  therapeutic  suspension  of  bacteria  which 
have  been  isolated  from  another  patient,  who  has  suf- 
fered from  a  similar  infection. 

Toxins. — The  soluble  products  of  bacterial  and  plant  growth, 
whereby  their  deleterious  effects  are  brought  about. 

Tuberculins. — Various  preparations  from  tubercle  bacilli  for 
therapeutic  and  diagnostic  purposes. 

Vaccination. — Protective  immunization  to  smallpox  by  in- 
oculation with  cow-pox  virus. 

Vaccine. — Term  properly  reserved  for  cow-pox  virus ;  now 
frequently  applied  to  therapeutic  suspensions  of  bacteria 
or  bacterins. 


INDEX 


Abderhalden-Fauser  reaction,  163 
Abderhalden  reaction,  157 

dialysis  method,  157 
optical  method,  160 
Pearce  and  "Williams'  modi- 
fication, 160 
Abscess,  bacterin  therapy  in,  262 

bronchial,    bacterin    therapy    in, 
291 
Absorption  of  complement,  90 
Acne  bacillus,  260 

bacterin  therapy  in,  260 
Acromegaly,  pituitary  gland  in,  315 
Actinomycosis,  bacterin  therapy  in,  267 
Actinomycotin,  267 

dosage,  251 
Adrenal  gland,  313 
Adrenalin,  314 

Adulteration  of  meat  products,  detec- 
tion of,  82 
Agglutination,  clinical  application  of, 

72,  81 
Agglutinins,  20,  72 
Alexins,  -i 

Allergic  reactions,  167 
Allergy,  18,  22 
Alopecia  areata,  bacterin  therapy  in, 

260 
Amboceptor,  26,  86 
Amenorrhoea,  corpus  luteum  in,  316 
Anaphylaxis,  23,  27,  167 
mechanism  of,  29 
passive,  31 

precautions  against,  32 
reaction  in  diagnosis,  31 
symptoms  of,  30 
Anti-anaphylaxis,  31 
Anti-anthrax  serum,  63 
Antibacterial  sera,  34,  55 

preparation  of,  38 
Antibody,  19 
Antibotulism  serum,  53 
Anticarcinomatous  extracts,  67 
Anticholera  serum,  62 
Anticolonic  serum,  61 
Antidiphtheritic    serum,    preparation 

of,  35 
Antidysenteric  serum,  52,  62 


Antifcrment,  22,  65 
Antigen,  8,  16,  89 

definition  of,  19 

titration  of,  111 
Antigonococcic  serum,  51,  59 
Antileprosy  serum,  64 
Antimelitensic  serum,  63 
Antimeningococcic  serum,  60 
Antiphytotoxic  serum,  53 
Antipneumococcic  serum,  58 
Antirabic  inoculation,  301 

serum,  64 
Antisera,  preparation  of,  34 

therapeutic  use  of,  40 
Antistaphylococcic  serum,  55 
Antistreptococcic  serum,  58 
Antitetanic  serum,  46 
Antithyroid  serum  and  extracts,  68 
Antitoxic  sera,  34,  42 

preparation  of,  35 

unit,  37 
Antitoxin,  20 

diphtheria,  42 

method  of  injection,  43 
preparation  of,  35 
protective  use  of,  42 

tetanus,  46 

therapeutic  use  of,  47 
Antituberculosis  serum,  52 
Antityphoid  extract  of  Jez,  65 

inoculation,  296 

serum,  61 
Antivenin,  53 
Arthigon,  276 
Arthritis,  bacterin  therapy  in,  281 

tubercuhn  in,  208 
Arthus'  phenomenon,  28 
Arylarsonates,  319 
Atoxyl,  319 

Aural  tuberculosis,  tuberculin  in,  209 
Auto-antibodies,  22 
Autogenous  bacterins,  224 
Auto-inoculation,  6 

induced,  244 
Autolysates,  7 
Autolysis  in  preparation  of  bacterins, 

221 
Autosalvarsanized  serum,  340 
353 


354 


INDEX 


Bacillus     acidi     lactici     in     bacterin 

therapy,  277,  293 
acnes  in  bacterin  therapy,  260 
anthracis  in  bacterin  therapy,  262, 

266 
coli  in  bacterin  therapy,  262,  266, 

273,  276,  277,  279,  280,  284,  290 
diphtherise  in   bacterin   therapy, 

285,  286 
dysenterise  in   bacterin   therapy, 

298 
fluorescens   in   bacterin  therapy, 

266,  284 
influenzae    in    bacterin    therapy, 

285,  286,  290,  292,  299 
Koch- Weeks  in  bacterin  therapy, 

283 
lactis      aerogenes      in      bacterin 

therapy,  266,  284 
lactis  bulgaricus  in  bacterin  ther- 
apy, 304 
mallei  in  bacterin  therapy,  262, 

268 
Morax  -  Axenfeld     in     bacterin 

therapy,  283 
of  Bordet-Gengou  in  bacterin  ther- 
apy, 292 
of  Friedlander  in  bacterin  therapy, 

273,  283 
pestis  in  bacterin  therapy,  271 
prodigiosus  in  bacterin  therapy, 

303 
proteus      vulgaris     in      bacterin 

therapy,  266,  284 
pseudodiphtherise      in      bacterin 

therapy,  285,  291 
pseudotuberculosis  rodentium  in 

bacterin  therapy,  273 
pyocyaneus  in  bacterin  therapy, 

262,  266,  277,  283,  284,  293 
tuberculosis  in  bacterin  therapy, 

262,  266,  273,  280,   283,  284, 

285,  292,  293 
typhosus  in  bacterin  therapy,  262, 

273,  277,  280,  285,  293 
Bacterisemia,  bacterin  therapy  in,  298 
Bacterial  infections,  natural  recovery 

from,  213 
Bacterial  inoculation,  216 

general  indications  for,  242 
Bacterial  suspensions,  standardization 

of,  219 
Bacterin  therapy,  accessory  measures 
in,  258 
causes  of  failure,  255 


Bacterin  therapy,  contra-indications, 
254 
in  various  diseases,  259-298 
limitations,  255 
opsonic  control  of,  225 
results  of,  259 

stock  versus  autogenous  bac- 
terins,  256 
Bacterins,  autogenous  vs.  stock,  224 
autolysis  of,  221 

clinical  symptoms  in  administra- 
tion of,  225 
containers  for,  223 
dosage  of,  250 
standardization  of,  219 
technic  of  administration  of,  248 
Bacteriocidin,  214 
Bacteriolysins,  21,  86 
Blood,  medico-legal  identification  of, 

83,  85 
Bordet-Gengou  phenomenon,  92 
Bubonic  plague,  bacterin  therapy  in, 
271 

Cancer,  inoculation  treatment  of,  302 
Carbunculosis,    bacterin    therapy    in, 

261 
Carcinoma,  meiostagmin  reaction  in, 
163 
sera  and  extracts  in,  67 
Cellulitis,  bacterin  therapy  in,  265 
Cerebrospinal     meningitis,      bacterin 

therapy  in,  300 
Chemotherapy,  318 
Cholera,  bacterin  therapy  in,  298 
Cholesterinizod   extracts    in    Wasser- 

mann  reaction,  96 
Coagulose,  307 
Coley's  fluid,  302 
Complement,  86 

fixation  reaction,  16 

gonococcus,  141 

in  echinococcus  disease, 

153 
in    proteid    differentia- 
tion, 155 
in  tuberculosis,  154 
in  typhoid  fever,  154 
Complement,  preparation  of,  116 

titration  of,  117 
Corneal  ulcer,  bacterin  therapy  in,  284 
Corpus  luteum,  316 
Corynebacterium     pseudodiphtheriti- 

cum  in  bacterin  therapy,  277 
Cretinism,  thyroid  in,  312 


INDEX 


355 


Crotalin,  69 

Cutaneous  reactions,  189 
Cystitis,  bacterin  therapy  in,  273 
Cytolysins,  21,  86 

Dermatitis,  bacterin  therapy  in,  265 
Diphtheria  antitoxin,  35,  42 

method  of  injection,  43 
preparation  of,  35 
protective  use  of,  42 
bacterin  therapy  in,  286 
carriers,  45,  288 
curative  treatment  of,  44 
prophylaxis,        von        Behring's 

method,  43 
toxic  skin  reaction,  191 
unit  of,  36 
Dose  table  of  bacterins,  251 
Dysentery,  bacterin  therapy  in,  298 
Dysmenorrhoea,  corpus  luteum  in,  316 
ovarian  substance  in,  316 

Echinococcus     disease,    complement- 
fixation  in,  153 
Ehrhch's  side-chain  theory,  24 
Endotoxins,  38 

EnterocoHtis,  bacterin  therapy  in,  293 
Epididymitis,  bacterin  therapy  in,  278 
Epilepsy,  crotalin  in,  69 
Epiphanin  reaction,  166 
Erysipelas,  bacterin  therapy  in,  265 
Exophthalmic  goitre,  thyroid  in,  313 
Eye  diseases,  bacterin  therapy  in,  283 

Fixation  of  complement,  90 
Furunculosis,  bacterin  therapy  in,  261 

Genito-urinary    diseases,     bacterin 
therapy  in,  273 
tuberculosis,  tuberculin  in,  208 
Glanders,  bacterin  therapy  in,  268 
Gonococcus  complement-fixation  test, 
141 
antigens,  143 
technic,  142 
in    bacterin    therapy,    275,    277, 
278-281,  283,  284 
Gonorrhoea,  cutaneous  reaction  in,  189 

Hsemocytometer  in  standardization  of 

bacterial  suspensions,  219 
Hemolysin,  natural,  91 
Hsemolysins,  21,  86 


Haemolysis,  in  transfusion  of  blood, 
309 
mechanism  of,  91 
non-specific,  90 
Haemolytic  amboceptor,   preparation 
of,  106 
titration  of,  108 
Hay  fever,  active  immunization  in, 

289 
Hecht- Weinberg  reaction,  130 
Hemorrhage,  serum  treatment  of,  306 

transfusion  of  blood  in,  308 
Horse  serum  in  treatment  of  hemor- 
rhage, 306 
Hydrophobia,  treatment  of,  300 
Hypersusceptibility,  27 
Hypophysis,  314 
Hypopyon,  bacterin  therapy  in,  284 

Identification  of  blood,  83,  85 
Immune  body,  20 
Immunity,  acquired,  5 

active,  duration  of,  247 

definition  of,  1 

local,  2 

mechanism  of,  8 

natural,  1 

passive,  duration  of,  41 
Immunization,  active,  5,  35 

passive,  8,  35 
Immunology,    history    and    develop- 
ment of,  10 
Impetigo,  bacterin  therapy  in,  265 
Infections,   bacterial,   recovery  from, 

213 
Inoculation,    bacterial,    general  indi- 
cations, 242 

therapeutic,  principles  of,  216  ^ 
Intestinal  tuberculosis,  tuberculin  in, 
•     208 

Iritis,  bacterin  therapy  in,  284 
Isocytolysins,  21 

Jarisch-Herxheimer  reaction,  345 
Jez,  antityphoid  extract  of,  65 

Kuhnhardt's  spreader,  233 

Labor,  pituitary  extract  in,  315 
Leprosy,  Wassermann  reaction  in,  132 
Leucocytic  extract,  65 
Ludwig's  angina,  bacterin  therapy  in, 

265 
Luetin  reaction,  186 


356 


INDEX 


Lymphadenitis,  bacterin  therapy  in, 
265 
tuberculous,  tuberculin  in,  209 
Lymphangitis,    bacterin    therapy    in, 

265 
Lysins,  21,  86 

Malaria,  Wassermann  reaction  in,  132 
Mallein,  268 

Meiostagmin  reaction,  17,  163 
Meningococcus   in   bacterin   therapy, 

300 
Menopause,  corpus  luteum  in,  316 

ovarian  extract  in,  316 
Micrococcus  albus  in  bacterin  therapy, 
260,  262,  277 
aureus  in  bacterin  therapy,  261, 

262,  266,  277 

candicans  in  bacterin  therapy,  277 

catarrhalis  in   bacterin   therapy, 

275-277,  283,  285,  286,  290,  292 

citreus  in  bacterin  therapy-,  262, 

277 
melitensis    in    bacterin    therapy, 

£99 
neoformans  in  bacterin  therapy, 
302 
Myxcedema,  thyroid  in,  312 

Neisser  bacterin,  275,  277-281 
Neisser-Sachs  reaction,  155 
Neosalvarsan,  chemical  formula,  321 

dosage,  326 

indications,  321 

preparation  of  solution,  338 

provocative  employment  of,  322 
Neurasthenia,  corpus  luteum  in,  316 
Noguchi  modification  of  Wassermann 
reaction,  129 

Obesity,  thyroid  in,  313 

Ocular  tuberculosis,  tuberculin  in,  209 

Opsonic  index,  15 

apparatus  for,  230 

definition  of,  229 

in  diagnosis    and    prognosis, 

229 
interpretation  of,  235 
in  tuberculin  therapy,  199 
limitations  of,  235 
negative  phase,  235 
positive  phase,  235 
preparation  of  smears,  233 
Simon's  method,  234! 
technic  of,  230 


Opsonic  index,  tuberculo-,  233 

value  of,  235 
Opsonins,  21,  215 

definition  of,  228 

immune,  22,  229 

normal,  22,  229 
Organotherapy,  312 
Osteitis,  bacterin  therapy  in,  280 

tuberculin  in,  208 
Osteomyelitis,    bacterin    therapy    in, 

280 
Osteophytes,  bacterin  therapy  in,  281 
Ovarian  substance,  316 

"  Paras\'philitic  "  affections  and  Was- 
sermann reaction,  136 

Paresis  and  Wassermann  reaction,  137 
treatment    by    autosaivarsanized 
serum,  341 

Passive  immunity,  duration  of,  41 

Pasteur  treatment  of  rabies,  300 

Pemphigus,  bacterin  therapy  in,  265 

Periostitis,  bacterin  therapy  in,  280 

Peritoneal  tuberculosis,  tuberculin  in, 
208 

Pertussis,  bacterin  therapy  in,  292 

Pfeiffer's  phenomenon,  87 

Phagoc3'tosis,  211 
spontaneous,  228 

Phylacogens,  70 

Pituitary  body,  314 

Pneumococcus  in  bacterin  therapy, 
262,  273,  276,  277,  279,  280,  282- 
284,  286,  291-293 

Pneumonia,  bacterin  therapy  in,  291 

Poisons,  difference  from  true  antigens, 
26 

Pollen  toxin  in  hay  fever,  289 

Precipitin  reaction,  82,  83 

Precipitins,  20 

Pregnancy,  Abderhalden's  test  for,  157 

Prostatitis,  bacterin  therapy  in,  277 

Protein  differentiation,  complement- 
fixation  in,  155 

Provocative  employment  of  salvarsan, 
322 

Puerperal  sepsis,  bacterin  therapy  in, 
280 

Pulmonary  tuberculosis,  tuberculin  in, 
206 

Pyaemia,  bacterin  therapy  in,  298 

Pyelitis,  bacterin  therapy  in,  273 

Pyelonephritis,  bacterin  therapy  in, 
273 

Pyocyanase,  68 


INDEX 


357 


Pyonephrosis,  bacterin  therapy  in,  273 
Pyorrhaa  alveolaris,  bacterin  therapy 
in,  292 

Rabies,  treatment  of,  300 
Reaction,  Abderhalden,  157 
Abderhalden-Fauser,  163 
Calmette,  185 

cutaneous,  in  gonorrhoea,  189 
in  tuberculosis,  180 
in  typhoid  immunity,  190 
Schick's     diphtheria     toxin, 
191 
epiphanin,  106 
gonococcus  complement-fixation, 

141 
Jarisch-Herxheimer,  345 
luetin,  186 
meiostagmin,  163 
Moro,  183 
Neisser-Sachs,  155 
precipitin,  82 

sero-enzyme,  in  mental  diseases, 
162 
in  pregnancy,  157 
in  syphilis,  161 
tuberculin,  173 
von  Pirquet,  180 
"VN'assermann,  95 
Widal,  73 
AVolff-Eisner,  185 
Receptors,  24 
Recovery    from    bacterial    infections, 

213 
Rhinitis,  bacterin  therapy  in,  286 

Salpingitis,  bacterin  therapy  in,  279 
Salvarsan,  acquired  resistance  to,  323 
chemico-physical  properties,  320 
clinical  reactionary  effects  of,  344 
contra-indications,  324 
dosage,  325 
excretion  of,  346 
history  of,  318 
indications,  321 
in  non-syphilitic  diseases,  322 
intramuscular  administration  of, 

327 
intraspinal  administration  of,  339 
intravenous  administration,  331 
after-care  of  patient,  343 
preparation  of  patient, 

332 
preparation  of  solution, 
333 


Salvarsan,    methods    of    preparation 
and  administration,  327 
precautions,  324 
provocative  employment  of,  143, 

322 
subcutaneous  administration,  328 
Sarcoma,  Coley's  fluid  in,  302 
Scarlet   fever,    AVassermann   reaction 

in,  132 
Schick's  diphtheria   toxin  skin   reac- 
tion, 191 
Seborrhea,  bacterin  therapy  in,  260 
Seminal  vesiculitis,  bacterin  therapy 

in,  277 
Septicaemia,  bacterin  therapy  in,  298 
Sera,  antibacterial,  34,  55 
preparation  of,  38 
antitoxic,  34 
Serobacterins,  222 

Sero-enzyme  reaction,  mental  diseases, 
162 
pregnancy,  157 
syphilis,  161 
Serum,  anti-anthrax,  63 
antibotulism,  53 
anticholera,  02 
an ti colonic,  61 
antidiphtheritic,  35,  42 
antidysenteric,  52,  62 
antigonococcic,  51,  59 
antileprosy,  64 
antimelitensic,  63 
antimeningococcic,  60 
antiphytotoxic,  53 
antipneumococcic,  58 
antirabic,  64 
antistaphylococcic,  55 
antistreptococcic,  58 
antitetanic,  46 
antithyroid,  68 
antituberculosis,  52 
antityphoid,  61 

normal,  in  treatment  of  hemor- 
rhage, 306 
sickness,  33 
Side-chain  theory,  24 
Simon's    method  for    opsonic    index, 

234 
Sinus,  bacterin  therapy  in,  266 
Sinusitis,  bacterin  therapy  in,  286 
Skin    diseases,    bacterin    therapy  in, 

259 
Smallpox  inoculation,  11 
vaccination,  268 
technic  of,  269 


358 


INDEX 


Staphylococcus    in  bacterin  therapy, 
262,   266,  273,  276,  279,  280, 
282-284,  286,  290,  292 
spray  for  diphtheria  carriers,  45 
Stock  bacterins,  224,  256 
Streptococcus    in    bacterin    therapy, 
262,  266,  273,  276,  277,  279, 
280,  282-284,  286,  290,  293 
rheumaticus,  281 
Streptothrix  actinomyces  in  bacterin 

therapy,  262,  291 
Swift-Ellis  treatment,  341 
Sycosis,  bacterin  therapy  in,  265 
Synovitis,  bacterin  therapy  in,  281 
Syphilis,  chemotherapy  in,  318 

latent,  and  Wassermann  reaction, 

134 
luetin  reaction  in,  186 
results  of  specific  treatment  in, 

348 
stages  of,  and  Wassermann  reac- 
tion, 133 


Tabes  and  Wassermann  reaction,  137 
treatment  of,  by  auto-salvarsan- 
ized  serum,  341 
Tetanus  antitoxin,  46 

therapeutic  use  of,  47 
Thymus  gland,  316 
Thyroid  gland,  312 
Tonsillitis,  bacterin  therapy  in,  293 
Toxin,  diphtheria,  36 
Toxins,  20 

Transfusion  of  blood,  308 
direct,  310 
indirect,  310 
TubercuUn,  168 

as  diagnostic  agent,  175 
Denys',  170 
Dixon's,  171 
dosage,  196 
focal  reaction,  175 
general  reaction,  174 
hypersusceptibility,  202 
local  reaction,  175 
modes  of  administration,  196 
new,  169,  170 
old,  168 

oral  administration,  198 
physiological  action  of,  173 
reaction,  Calmette,  185 
Detre,  182 
intradermic,  180 
Moro,  183 


Tuberculin    reaction,   mucous   mem- 
brane instillation,  185 
percutaneous,  183 
scarification,  180 
subcutaneous,  176 
von  Pirquet,  180 
Wolff-Eisner,  185 
rectal  administration,  199 
Spengler's,  171 
subcutaneous  injection,  176 
technic  of  making  dilutions,  172 
therapeutic  administration,  194 
therapy,  193 

available  preparations,  195 
clinical  symptomatology^  199 
control  of,  199 
indications  and  results,  205 
in  various  diseases,  206-209 
limitations  and  contra-indica- 

tions,  203 
mixed  infection  and,  205 
opsonic  control  of,  199 
Tuberculinum  purum,  172 
Tuberculo-opsonic  index,  233 
Tuberculosis,  aural,  tuberculin  in,  209 
bone  and  joint,  tuberculin  in,  208 
complement-fixation  in,  154 
genito-urinary,  tuberculin  in,  208 
intestinal  and  peritoneal,  tubercu- 
lin in,  208 
ocular,  tuberculin  in,  209 
of   lymph-nodes,    tuberculin    in, 

209 
prophylaxis,  193 
pulmonary,  tubercuUn  in,  206 
Trypanosomiasis     and     Wassermann 

reaction,  132 
Typhoid  carriers,  treatment  of,  295 
bacterin  therapy  in,  294 

complement-fixation  in,  154 
immunity,  cutaneous  reaction  in, 
190 

Ulcer,  corneal,  bacterin  therapy  in,  284 
Ulcers,  bacterin  therapy  in,  263 
Urethritis,  bacterin  therapy  in,  263 
Uterine  inertia,  pituitary  extract  in, 

315 
Uveitis,  bacterin  therapy  in,  284 

Vaccination  against  smallpox,  11,  268 
Vaccinia,  characteristics  of,  270 
Vaccinoid,  270 
Variola,  268 


INDEX 


359 


Vibrio  cholerae  in  bacterin  therapy, 

298 
Von  Behring's  method  of  prophylaxis 

in  diphtheria,  43 
Von  Pirquet  reaction,  180 
Vulvovaginitis,   bacterin   therapy  in, 

279 

TN'assermann  reaction,  95 

alcohohsm  and,  135 
antigens  in,  96 
apparatus  for,  102 
cholesterinized  extracts  in,  96 
chnical  apphcation  of,  131 
collection  of  patient's  serum, 

114 
effects  of  treatment  on,  138 
htemolytic  system,  103 
Hccht  -  Weinberg   modifica- 
tion, 130 
inactivation      of      patient's 

serum,  121 
in  framboesia,  132 
in  inlierited  sjphilis,  136 
in  latent  syphilis,  134 
in  leprosy,  132 
in  malaria,  132 
in   parasvphilitic   affections, 

135 
in  paresis,  137 
in  scarlet  fever,  132 
in  tabes,  137 


Wassermann   reaction  in  trypanoso- 
miasis, 132 
in  yaws,  132 
modifications  of,  128 
Noguchi  modification  of,  129 
preparation   of  amboceptor, 

106 
preparation  of  complement, 

116 
preparation  of  corpuscle  sus- 
pension, 116 
provocative  treatment,  140 
quantitative,  125 
reading  of  results,  123 
sheep's  blood  in,  103 
technic  of,  102 
titration  of  antigen.  111 
of  complement,  117 
Whooping-cough,  bacterin  therapy  in, 

292 
Widal  reaction,  73 

macroscopic  method,  79 
microscopic  method,  73 
significance  of,  80 
technic  of,  77 
use  of  dried  blood  in,  76 
Wright's  capsule,  74,  231 

method    of    standardization    of 
bacterial  suspensions,  220 


Yaws  and  W^assermann  reaction. 
Yeast,  304 


132 


AN     INITIAL     FINE     OF    25     CENTS 

WILL  BE  ASSESSED  FOR  FAILURE  TO  RETURN 
THIS  BOOK  ON  THE  DATE  DUE.  THE  PENALTY 
WILL  INCREASE  TO  50  CENTS  ON  THE  FOURTH 
DAY  AND  TO  $1.00  ON  THE  SEVENTH  DAY 
OVERDUE. 


NOV  23  1932 

DEC  6    1938 
JUL  2  3  1943 


b 


LD  21-50m-8,32 


U.C.  BERKELEY  LIBRARIES 


CDMDDME3fl3 


308651   ^jsy]7^f 


UNIVERSITY  OF  CALIFORNIA  LIBRARY 


